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University of California • Berkeley

Regional Oral History Office University of California

The Bancroft Library Berkeley, California

Program in the History of the Biosciences and Biotechnology

Horace A. Barker, Ph.D.
SCIENTIST AND PROFESSOR OF MICROBIAL BIOCHEMISTRY AT BERKELEY

With an Introduction by
Clinton E. Ballou, Ph.D.

Interviews Conducted by

Sally Smith Hughes, Ph.D.

in 1998 and 1999

Since 1954 the Regional Oral History Office has been interviewing leading
participants in or well-placed witnesses to major events in the development of
northern California, the West, and the nation. Oral history is a method of
collecting historical information through tape-recorded interviews between a
narrator with firsthand knowledge of historically significant events and a well-
informed interviewer, with the goal of preserving substantive additions to the
historical record. The tape recording is transcribed, lightly edited for
continuity and clarity, and reviewed by the interviewee. The corrected
manuscript is indexed, bound with photographs and illustrative materials, and
placed in The Bancroft Library at the University of California, Berkeley, and in
other research collections for scholarly use. Because it is primary material,
oral history is not intended to present the final, verified, or complete
narrative of events. It is a spoken account, offered by the interviewee in
response to questioning, and as such it is reflective, partisan, deeply involved,
and irreplaceable.

********************** **************

All uses of this manuscript are covered by a legal agreement
between The Regents of the University of California and Horace M.
Barker dated January 14, 1999. The manuscript is thereby made
available for research purposes. All literary rights in the
manuscript, including the right to publish, are reserved to The
Bancroft Library of the University of California, Berkeley. No part
of the manuscript may be quoted for publication without the written
permission of the Director of The Bancroft Library of the University
of California, Berkeley.

Requests for permission to quote for publication should be
addressed to the Regional Oral History Office, 486 Bancroft Library,
Mail Code 6000, University of California, Berkeley 94720-6000, and
should include identification of the specific passages to be quoted,
anticipated use of the passages, and identification of the user.
The legal agreement with Horace M. Barker requires that he be
notified of the request and allowed thirty days in which to respond.

It is recommended that this oral history be cited as follows;

Horace M. Barker, Ph.D., "Scientist and
Professor of Microbial Biochemistry at
Berkeley," an oral history conducted in
1998 and 1999 by Sally Smith Hughes,
Ph.D., Regional Oral History Office, The
Bancroft Library, University of
California, Berkeley, 2001.

Copy no.

Horace Albert Barker

New York Times. January 10,
2001.

Horace Barker, 93, Scientist
Who Studied Body Chemistry

By WOLFGANG SAXON

Dr. Horace Albert Barker, a mi-
crobiologist and biochemist who
helped to unravel the complex pro
cesses of chemical conversion inside
living organisms, died on Dec. 24 at
his home in Berkeley, Calif. He was
93.

Dr. Barker made his mark in the
1950's by tracing the biochemical
function of vitamin B12. His investi
gation helped explain complex ways
chemistry works in the body.

Earlier, in 1944, he was on a team
of researchers that detected the role
of enzymes when living cells synthe
size sucrose. The researchers gained
that insight with one of the earliest
laboratory uses of radioactive car-
bon-14 tracers, a technique Dr. Bark
er helped pioneer.

For his work, specifically that in
volving vitamin B12, Dr. Barker re
ceived one of the 12 National Medals
of Science for 1968 from President
Lyndon B. Johnson.

Born in Oakland, Calif., Horace
Barker graduated in 1929 from Stan
ford University, where he also re
ceived a Ph.D. in chemistry four
years later.

Dr. Barker became an instructor
in soil microbiology at the University
of California in 1936. He was named a
professor in the department of bio
chemistry when it was set up in 1959

Jane Scherr. 1992

', Dr. Horace A. Barker

and served as chairman in the 1960's.

Dr. Barker's studies dealt with vi
tamin B12 coenzymes, vitamin
chemistry, bacterial metabolism,
fatty acid oxidation and synthesis,
carbohydrate transformations, and
amino acid and purine metabolism.
Together, the studies helped build a
foundation for much of what is
known today of metabolism and its
role in sickness and health.

Working with a common soil bac
terium dredged from the mud of San
Francisco Bay, Dr. Barker led a re
search team that in 1959 discovered
vitamin B12 coenzyme, an active
form of vitamin B12, deployed in
vital chemical conversions in the
body. He then mapped out many of
the metabolic reactions this entails.

In doing so, Dr. Barker clarified
the vitamin B12 coenzyme's role in
building body tissue. This in turn
contributed to the understanding of
several diseases, including perni
cious anemia caused by a deficiency
of vitamin B12.

Dr. Barker wrote or helped write
some 230 scientific publications.
Among his honors was his election to
the National Academy of Sciences
and to the American Academy of
Arts and Sciences.

Dr. Barker is survived by two'
daughters, Barbara B. Friede, of
Piedmont, Calif., and Elizabeth F.
Mark, of Lexington, Mass.; a son,
Robert H., of Camino, Calif.; and
four grandchildren. His wife, Marga
ret McDowell Barker, died in 1995
after 62 years of marriage.

Dr. Barker retired with emeritus
status in 1975 but remained active in
the biochemistry department at
Berkeley well after turning 80.

Cataloguing information

Horace A. Barker, Ph.D. (1907-2000) Professor of Biochemistry

Scientist and Professor of Microbial Biochemistry at Berkeley, 2001, xix,
118 pp.

Stanford University, thesis in chemistry, interest in botany; work with
Cornelius van Niel, Hopkins Marine Station; fellow, Microbiology Laboratory,
Polytechnical School, Delft, Holland; Berkeley, 1936-75: soil microbiologist,
Agriculture Experiment Station, 1936-50; Dept. of Biochemistry, 1950-75:
discusses program in Comparative Biochemistry, Dept. of Bacteriology, Virus
Laboratory; research: photosynthetic bacteria, enzymatic synthesis of sucrose,
B12 coenzyme, use of radioactive tracers from Berkeley cyclotron, patent on B12
process; comments on scientists: C. B. van Niel, A. J. Kluyver, Sam Ruben,
Martin Kamen, Ernest and John Lawrence, Wendell Stanley, and others.

Introduction by Clinton E. Ballou, Ph.D., Professor Emeritus,
Biochemistry, UC Berkeley.

Interviewed 1998-1999 by Sally Smith Hughes for the Program in the
History of Biosciences and Biotechnology, Regional Oral History Office,
The Bancroft Library, University of California, Berkeley, 2001.

TABLE OF CONTENTS- -Horace A. Barker

SERIES HISTORY by Sally Smith Hughes i

INTRODUCTION by Clinton E. Ballou vii

INTERVIEW HISTORY by Sally Smith Hughes xvi

BIOGRAPHICAL INFORMATION xix

I HIGHER EDUCATION, 1926-1936 1
Stanford University, 1926-1933 1

Research Assistant with C. V. Taylor, 1930-1931 1

Thesis in the Chemistry Department 2

Early Interest in Botany 3

Cornelius van Niel and the Hopkins Marine Station, Pacific

Grove, California, 1931-1935 5

Van Niel as a Personality 5

Work with J. P. Baumberger, Summer 1931 5

Working with van Niel 6

Van Niel's Microbiology Course 7

Rockefeller Foundation Fellow, Microbiology Laboratory,

Polytechnical School, Delft, 1935-1936 8
Physical Layout and Operation of the Microbiology

Laboratory 8

Research on Methane-producing Bacteria 10

The Enrichment Culture Technique 11

II BIOCHEMIST AT BERKELEY, 1936-1975 12
Faculty Member and Soil Microbiologist, UCB Agriculture

Experiment Station, 1936-1950 12

First Position at Berkeley 12

Early Faculty Members in the Berkeley Agricultural Program 13

Teaching 16

Early Courses 16

The Program in Comparative Biochemistry 18

Schism in the Department of Bacteriology 19

Research with Radioactive Tracers 20

Collaborations with Sam Ruben and Martin Kamen 20

Early Tracer Experiments 23

Wendell Stanley 26

Interaction with Campus Groups Doing Biochemistry 27

The Delft Laboratory of Microbiology 29

Orientation towards Microbial Biochemistry and Natural

History 29

Physical Layout and Personnel 30

Dr. Barker's Research 32

More on Faculty Participants 51

Student Participation 52

Seminars 54

Teaching 56

Course in Soil Microbiology 56

The van Niel Approach to Biochemistry 56

Barker's Teaching Style 58

Research on Anaerobic Bacteria 59

TAPE GUIDE 60

APPENDIX 61

H. A. Barker correspondence, B-12 research 62

U.S. Patent 3,037,016 66
"Explorations of Bacterial Metabolism," H. A. Barker, Ann. Rev.

Biochem. 1978. 47:1-33 75

Horace A. Barker, Curriculum Vitae 108

Obituary, UC Berkeley Media Release, January 8, 2001 110

Memorial Service Program 113

INDEX 117

BIOTECHNOLOGY SERIES HISTORY--Sally Smith Hughes, Ph.D.

Genesis of the Program in the History of the Biological Sciences and
Biotechnology

In 1996, a long-held dream of The Bancroft Library came true with the
launching of its Program in the History of the Biological Sciences and
Biotechnology. For years, Bancroft had wished to document the history of
the biological sciences on the Berkeley campus, particularly its
contributions to the development of molecular biology. Bancroft has strong
holdings in the history of the physical sciences — the papers of E.O.
Lawrence, Luis Alvarez, Edwin McMillan, and other campus figures in physics
and chemistry, as well as a number of related oral histories. These
materials support Berkeley's History of Science faculty, as well as
scholars from across the country and around the world.

Although the university is located next to the greatest concentration
of biotechnology companies in the world, Bancroft had no coordinated
program to document the industry nor its origins in academic biology. For
a decade, the staff of the Regional Oral History Office had sought without
success to raise funds for an oral history program to record the
development of the industry in the San Francisco Bay Area. When Charles
Faulhaber arrived in 1995 as Bancroft's new director, he agreed to the need
to establish a Bancroft program to capture and preserve the collective
memory and papers of university and corporate scientists and the pioneers
who created the biotechnology industry. He too saw the importance of
documenting the history of a science and industry which influences
virtually every field of the life sciences, generates constant public
interest and controversy, and raises serious questions of public policy.
Preservation of this history was obviously vital for a proper understanding
of science and business in the late twentieth century.

Bancroft was the ideal location to launch such an historical
endeavor. It offered the combination of experienced oral history and
archival personnel, and technical resources to execute a coordinated oral
history and archival program. It had an established oral history series in
the biological sciences, an archival division called the History of Science
and Technology Program, and the expertise to develop comprehensive records
management plans to safeguard the archives of individuals and businesses
making significant contributions to molecular biology and biotechnology.
It also had longstanding cooperative arrangements with UC San Francisco and
Stanford University, the other research universities in the San Francisco
Bay Area. The history of biotech project was to provide a basis for
continuing collaboration among the three institutions in the documentation
of recent science and technology through oral history and archival
collection. The only ingredient missing was funding.

In April 1996, the dream became reality. Daniel E. Koshland, Jr.
provided seed money for a center at The Bancroft Library for historical
research on the biological sciences and biotechnology. Thanks to this
generous gift, Bancroft has begun to build an integrated collection of
research materials—primarily oral history transcripts, personal papers,
and archival collections—related to the history of the biological sciences
and biotechnology in university and industry settings. One of the first
steps was to create a board composed of distinguished figures in academia
and industry who advise on the direction of the oral history and archival
components. The Program's initial concentration is on the San Francisco
Bay Area and northern California. But its ultimate aim is to document the
growth of molecular biology as an independent field of the life sciences,
and the subsequent revolution which established biotechnology as a key
contribution of American science and industry.

UCSF Library, with its strong holdings in the biomedical sciences, is
a collaborator on the archival portion of the Program. David Farrell,
Bancroft's curator of the History of Science and Technology, serves as
liaison. In February 1998, Robin Chandler, head of UCSF Archives and
Special Collections, completed a survey of corporate archives at local
biotechnology companies and document collections of Berkeley and UCSF
faculty in the biomolecular sciences. The ultimate aim is to ensure that
personal papers and business archives are collected, cataloged, and made
available for scholarly research.

Project Structure

With the board's advice, Sally Hughes, a science historian at the
Regional Oral History Office, began lengthy interviews with Robert Swanson,
a co-founder and former CEO of Genentech in South San Francisco; Arthur
Kornberg, a Nobel laureate at Stanford; and Paul Berg, also a Stanford
Nobel laureate. A short interview was conducted with Niels Reimers of the
Stanford and UCSF technology licensing offices. These oral histories build
upon ones conducted in the early 1990s, under UCSF or Stanford auspices,
with scientists at these two universities.1 The oral histories offer a
factual, contextual, and vivid personal history that enriches the archival
collection, adding information that is not usually present in written
documents. In turn, the archival collections support and provide depth to
the oral history narrations.

'Hughes conducted oral histories with Herbert Boyer, William Rutter,
and Keith Yamamoto of UCSF, and with Stanley Cohen of Stanford. To date,
the first volume of the oral history with Dr. Rutter is available at the
Bancroft and UCSF libraries; transcripts of the other interviews are
currently under review by the interviewees.

Ill

Primary and Secondary Sources

This oral history program both supports and is supported by the
written documentary record. Primary and secondary source materials provide
necessary information for conducting the interviews and also serve as
essential resources for researchers using the oral histories. The oral
histories also orient scholars unfamiliar with the field or the scientist
to key issues and participants. Such orientation is particularly useful to
a researcher faced with voluminous, scattered, and unorganized primary
sources. This two-way "dialogue" between the documents and the oral
histories is essential for valid historical interpretation.

Beginning with the first interviews in 1992, the interviewer has
conducted extensive documentary research in both primary and secondary
materials. She gratefully acknowledges the generosity of the scientists
who have made their personal records available to her: Paul Berg, Stanley
Cohen, Arthur Kornberg, William Rutter, and Keith Yamamoto. She also
thanks the archivists at Bancroft, UCSF, and Stanford libraries, and
personnel at Chiron, Genentech, and Stanford's Office of Technology
Licensing, for assistance in using archival collections.

Oral History Process

The oral history methodology used in this program is that of the
Regional Oral History office, founded in 1954 and producer of over 1,600
oral histories. The method consists of research in primary and secondary
sources; systematic recorded interviews; transcription, light editing by
the interviewer, and review and approval by the interviewee; library
deposition of bound volumes of transcripts with table of contents,
introduction, interview history, and index; cataloging in UC Berkeley and
national online library networks (MELVYL, RLIN, and OCLC) ; and publicity
through ROHO news releases and announcements in scientific, medical, and
historical journals and newsletters and via the ROHO and UCSF Library Web
pages.

Oral history as a historical technique has been faulted for its
reliance on the vagaries of memory, its distance from the events discussed,
and its subjectivity. All three criticisms are valid; hence the necessity
for using oral history documents in conjunction with other sources in order
to reach a reasonable historical interpretation.1 Yet these acknowledged
weaknesses of oral history, particularly its subjectivity, are also its
strength. Often individual perspectives provide information unobtainable
through more traditional sources. Oral history in skillful hands provides
the context in which events occur-- the social, political, economic, and

'The three criticisms leveled at oral history also apply in many cases
to other types of documentary sources.

institutional forces which shape the course of events. It also places a
personal face on history which not only enlivens past events but also helps
to explain how individuals affect historical developments.

An advantage of a series of oral histories on a given topic, in this
case molecular biology and biotechnology, is that the information each
contains is cumulative and interactive. Through individual accounts, a
series can present the complexities and interconnections of the larger
picture. Thus the whole (the series) is greater than the sum of its parts
(the individual oral histories), and should be considered as a totality.

Emerging Themes

Although the oral history program is still in its infancy, several
themes are emerging. One is "technology transfer," the complicated process
by which scientific discovery moves from the university laboratory to
industry where it contributes to the manufacture of commercial products.
The oral histories show that this trajectory is seldom a linear process,
but rather is influenced by institutional and personal relationships,
financial and political climate, and so on.

Another theme is the importance of personality in the conduct of
science and industry. These oral histories testify to the fact that who
you are, what you have and have not achieved, whom you know, and how you
relate has repercussions for the success or failure of an enterprise,
whether scientific or commercial. Oral history is probably better than any
other methodology for documenting these personal dimensions of history.
Its vivid descriptions of personalities and events not only make history
vital and engaging, but also contribute to an understanding of why
circumstances occurred in the manner they did.

Molecular biology and biotechnology are fields with high scientific
and commercial stakes. As one might expect, the oral histories reveal the
complex interweaving of scientific, business, social, and personal factors
shaping these fields. The expectation is that the oral histories will
serve as fertile ground for research by present and future scholars
interested in any number of different aspects of this rich and fascinating
history.

Update. September 2001

In early 2001, the Program in the History of the Biological Sciences
and Biotechnology was given great impetus by Genentech's generous pledge of
one million dollars to support documentation of the biotechnology industry.
At an initial meeting of Genentech and Library personnel in November 2000,
it was agreed that the initial phase of the Genentech-supported project in
the company's twenty-fifth anniversary year should focus on oral histories

with current and former Genentech employees. Archival collection, on the
other hand, was designated as a long-term process because of the greater
necessity to gather oral documentation while minds are clear and because of
Genentech 's present need to retain many corporate documents for legal and
other reasons .

On October 15, 2001, The Bancroft Library will celebrate Genentech' s
twenty- fifth anniversary and acknowledge its generosity to the Program by
formally presenting the oral histories of Herbert W. Boyer and Robert A.
Swanson, the company's founders. Oral histories are currently in progress
with the following individuals presently or formerly at Genentech: David
Goeddel, Arthur Levinson, Fred Middleton, Richard Scheller, and Daniel
Yansura. Oral histories are also completed or in progress with individuals
at Chiron Corporation and Tularik, Inc. The next phase will expand
documentation to other biotechnology companies.

Location of the Oral Histories

Copies of the oral histories are available at the Bancroft, UCSF, and
UCLA libraries. They also may be purchased at cost through the Regional
Oral History Office. Some of the oral histories, with more to come, are
available on The Bancroft Library's History of the Biological Sciences and
Biotechnology Website: http://www.lib.berkeley.edu/BANC/Biotech/.

Sally Smith Hughes, Ph.D.
Historian of Science

Regional Oral History Office
The Bancroft Library
University of California, Berkeley
October 2001'

Program in the History of the Biological Sciences and Biotechnology

Completed Oral Histories

November 2001

Horace A. Barker, Scientist and Professor of Microbial Biochemistry at
Berkeley, 2001

Paul Berg, Ph.D., A Stanford Professor's Career in Biochemistry, Science
Politics, and the Biotechnology Industry, 2000

Herbert W. Boyer, Ph.D., Recombinant DNA Research at UCSF and Commercial
Application at Genentech, 2001

Arthur Kornberg, M.D., Biochemistry at Stanford, Biotechnology at DNAX, 1998

Niels Reimers, Stanford's Office of Technology Licensing and the Cohen /Boyer
Cloning Patents, 1998

William J. Rutter, Ph.D., The Department of Biochemistry and the Molecular

Approach to Biomedicine at the University of California, San Francisco,
Volume I, 1998

Robert A. Swanson, M.S., Co-Founder, CEO, and Chairman of Genentech, Inc.,
1976-1996, 2001

Oral Histories in Process

Stanley N. Cohen, M.D.

David Goeddel, Ph.D.

Dennis Kleid, Ph.D.

Daniel E. Koshland, Ph.D.

Marian E. Koshland, Ph.D., retrospective

Arthur Levinson, Ph.D.

Fred Middleton

Thomas Perkins

Reorganization of Biology at UC Berkeley

William Rutter, Ph.D., Volume II

Richard Scheller, Ph.D.

vii
INTRODUCTION by Clinton E. Ballou, Ph.D.

Professor H. A. Barker was born on November 29, 1907, in Oakland,
California, where he grew up not far from Lake Merritt. At an early
age, he was given the nickname "Nook" by family members, who saw a close
resemblance to a popular cartoon character with a similar name (1).
This nickname stayed with him all his life and was adopted widely by
friends and colleagues. Later, in official correspondence, he signed
his name "Horace," although on less formal documents he occasionally
used "Al," a variant of his middle name. He was introduced to his
favorite avocation at an early age, when he went trout fishing on some
of the small streams that flowed from the Oakland and Berkeley Hills
into San Francisco Bay (2), and this love for nature and the outdoors
lasted throughout his life. When he was eleven, the family moved to
Palo Alto where his father, Albert C. Barker, was a teacher and school
administrator (3). Before their marriage, both of his parents had
attended Stanford University, where his future mother, Nettie Hindry,
obtained degrees in classical literature and Latin, and it is possible
that the name Horace reflected her classical interests.

Barker has noted that "Both my father and mother were very fond of
the outdoors and so each summer we spent a month or more, whenever
possible, camping in the Sierras, and living a quiet and simple life in
close contact with Nature. This resulted in my developing a
considerable familiarity with plants and animals, and the physical
environment, and perhaps even more important, developing a sense of
satisfaction and accomplishment in relatively solitary activities such
as fishing, hiking, and exploring new areas; this attitude was easily
carried over to scientific work in a laboratory." (3) He also developed
an interest in music, played the piano, and, after graduation from high
school, spent a year in Germany with his family where he "learned
German, read classical German literature, and went to innumerable operas
and concerts of every kind." (3) In 1925, he entered Stanford
University, obtained an undergraduate degree in physical sciences, and
left there in 1933 with a Ph.D. in Chemistry. While at Stanford, he
also met and married Margaret D. McDowell, with whom he had three
children, Barbara Freide of Piedmont, California, Betsy Mark of
Lexington, Massachusetts, and Bob Barker of Camino, California.

In his oral history, it is made clear that the three years
following his graduation from Stanford were a formative time in
stimulating his interest in microbiology and then concentrating his
attention on microbial biochemistry as the focus of his academic career.
In 1933, Barker set out on a two-year fellowship to study with C. B.
van Niel at the Hopkins Marine Station on Monterey Bay. He was the first
postdoctoral student of this young Stanford assistant professor who was
to become an icon of microbiology and a magnet that, over several

viii

decades, attracted many scientists with an interest in microbiology to
enroll in his famous summer course at the station. Although Barker had
developed some interest in biology as an undergraduate, particularly
botany, it was at Pacific Grove that he really committed to the subject.
He once recalled with awe how he would meet regularly with van Niel for
a one-on-one lecture, given entirely without notes, that might run for
an hour or more while delving deeply into some current topic. (3)

Barker also credited van Niel with introducing him to the valuable
technique of enrichment culture for the isolation of microorganisms
capable of effecting almost any desired biochemical reaction. I was a
beneficiary of this indoctrination when, in the 1960s, my research on
the structure of yeast mannans was stymied for lack of enzymes that
could selectively degrade the carbohydrate chains. In frustration, I
sought the council of then Professor Barker, who advised me to get some
rich dirt, put it in a test tube with an aqueous solution of mannan, and
wait until something grew up. Any microorganism that grew must be able
to hydrolyze the polysaccharide to give free mannose, a sugar similar to
glucose, that the organism could use as an energy source. Success
followed success with several different mannans, and we soon had a
collection of bacterial strains for the isolation of different
mannosidases (enzymes) with which we could take the polysaccharide apart
in a selective and stepwise manner.

When his fellowship at the Marine Station ended in 1935, Barker
backtracked on the footsteps of his mentor van Niel to study for a year
with A. J. Kluyver at the Delft Microbiology Laboratory. It was during
this year in Holland that he initiated an investigation that many years
later would lead him to the important discovery of vitamin B-12
coenzymes. (3) Also, during this year Barker received an invitation
from the University of California at Berkeley to join the Agriculture
Experiment Station as an instructor in soil microbiology, an opportunity
he attributed to van Niel's intercession. Two other promising young
biologists, William Zev Hassid (in plant nutrition) and Michael
Doudoroff (in bacteriology), accepted Berkeley appointments during the
same era, and later these three were to become closely associated in
teaching and research, with results that would bring distinction to the
campus .

In his oral history, Barker emphasized events prior to 1950, when
he was getting started at Berkeley, and perhaps these years were the
most memorable to him because they had such an influence on his later
career. It would be unfortunate, however, for anyone reading the
history to come away without a more complete picture of the man and his
role in science. To bring this picture into focus, it is helpful to
list some of the outstanding scientists who as students gained their
training with Barker, who worked as visitors in his laboratory, or with
whom he had an important association. These include Earl and Thressa
Stadtman, Fritz Lipmann, Eugene Kennedy, Joseph Wachsman, Irwin
Gunsalus, Arthur Kornberg, Jesse Rabinowitz, Herbert Weissbach, Harry

Hogenkamp, Benjamin Volcani, Roscoe Brady, Gerhard Gottschalk, Bernard
Horecker, Ralph Costilow, Robert Switzer, Robert Blakeley, Ching C.
Wang, and Ernst Winnacker. (4) Thus, Barker was mentor to a number of
young scientists who went on to outstanding careers, and he attracted
many distinguished investigators from around the world to his laboratory
in Berkeley.

Barker's major research activities dealt with studies on anaerobic
fermentation by bacteria. He elucidated a general pathway in bacteria
for the formation of methane from carbon dioxide, acetate and methanol,
and in so doing he was the first to demonstrate (with Sam Ruben and
Martin D. Kamen) the use of the long-lived radioactive isotope carbon- 14
"as a tracer in a biological system." (5) Then, using similar
techniques, he demonstrated the reductive incorporation of carbon
dioxide and of ethanol into short-chain fatty acids and various amino
acids. Turning his attention to the bacterial fermentation of amino
acids, he uncovered new pathways for their decomposition that, with
glutamate as a substrate, involved a novel chain rearrangement. This
reaction was found to be dependent on vitamin B-12, which led Barker to
the isolation and partial characterization of the coenzyme forms of the
vitamin, reported in 1960. A detailed account of these and other
studies is given in (3). In 1964, the British chemist Dorothy Hodgkin
was awarded the Nobel Prize for her work on vitamin B-12 structure, and
many of his colleagues feel that Barker could have shared in the prize.
This view was supported recently by Professor J. R. Quayle, F.R.S., who
observed that, "Looking back at Barker's overall achievements there is
no doubt that they are world-class, at the Nobel level. He entered the
field of bacterial fermentations when mixed cultures were the order of
the day. He and [Robert] Hungate developed pure culture isolation
techniques to the point that many people came to Berkeley to find out
how. Barker's intuition and meticulous analytical approach reduced
complex fermentations into a series of intellectually elegant equations
and carbon balances. In this he could be matched by few. He entered
the methanogenesis field in its prehistoric state and, again, provided
cultures and a chemical rationale that guided workers in the field for
years to come." (6)

In unpublished notes prepared by Barker in 1969 (5), the following
two sentences appear. "Before coming to Berkeley in September 1936, I
had investigated the biological formation of methane from ethanol,
acetate and butyrate and had obtained evidence for the theory of C. B.
van Niel that methane is formed by reduction of carbon dioxide. In the
fermentation of ethanol in the presence of calcium carbonate by
enrichment cultures of methane bacteria, I found that one mole of
carbonate was reduced to methane for each two moles of ethanol oxidized
to acetate." Here we see stated the topic that would occupy Barker for
much of his career as he sought to define the biochemical mechanisms
involved in such a seemingly simple transformation. Although he was
taken on several side journeys along the way, finding the mechanisms and
pathways in bacteria by which carbon dioxide was utilized as an oxidant,

by which methane was produced, and by which various other products
resulted from the fermentation of ethanol and amino acids, would consume
most of his energy for the years to come.

Also contained in one of these sentences is a hint of the special
characteristic that defined Barker's approach to science. He was
careful in designing his experiments and meticulous in accounting for
the stoichiometry of the reactants and products in any investigation.
Because of this practice, he was led to insights that might have eluded
the less attentive investigator. Thus, when he wrote, "I found that one
mole of carbonate was reduced to methane for each two moles of ethanol
oxidized to acetate," (5) one could rely on this as being significant
and near to the truth. Sometimes, however, he appeared to carry this
concern for numbers and accountability to an extreme. One cold winter
day in December, we were taking a trip together by car to fish for
steelhead on the Eel River, and Barker was driving. He decided to stop
for gas, and I was surprised when he withdrew a small black notebook
from the glove compartment and recorded the date and the exact mileage
on the speedometer, along with the amount of gas purchased and the cost.
I also saw that it was a well-used book that contained page after page
with columns of similar figures, and I have long pondered his attention
to such detail. I should add that this fishing trip also revealed
another characteristic of Barker, namely the persistent determination
with which he approached a problem. On this day, the problem was to
catch a fish, and steelhead are not the easiest quarry to pursue
successfully. After about eight hours of fruitless casting on that cold
and blustery day, I had given up, but Barker continued on until, when
the rest of us had reeled in our lines and were ready to leave, he set
the hook and eventually landed a seven-pound beauty. Mission
accomplished .

In his daily approach to science, Barker was somewhat detached and
completely unperturbable. He did not concern himself with the real or
imagined threat from competitors that motivates many scientists.
Perhaps he sought consciously to avoid research projects where the
competition was extreme. On one occasion, however, when his lab was
zeroing in on the B-12 coenzymes, he was faced with such a situation.
To learn first-hand how he acted at the time, I asked Herbert Weissbach,
a visiting scientist from the NIH and Barker's main collaborator on this
project from 1958-60, to share his experience. He wrote (7) "My year
with Barker was truly unforgettable. I would not say he was 'laid-back1
but he certainly had complete control of his emotions .. .the day we
showed the unknown cofactor for the conversion of glutamate to beta
methyl aspartate was a derivative of vitamin B-12. I had taken this
orange solution which had a spectrum that was not similar to anything
known and, after exposing it to light, the color changed and the new
spectrum was that of hydroxy B-12. This must have been in July or
August, on the day before Nook was to leave for his vacation home. I
came running into his office with what I thought was exciting news and
he said that the results were very nice. I suggested that we discuss

what experiments to do and put together a manuscript as quickly as
possible. He just continued what he was doing and said it could wait
until after he came back from vacation. Having come from the NIH, where
in a similar situation the manuscript would have been written that day,
I was shocked to say the least. I remember meeting Esmond Snell in the
hall and telling him about Nook's reaction, which to my amazement didn't
surprise him at all."

In the 1940s, Barker, Doudoroff, and Hassid were located in
neighboring labs on the third floor of the Life Sciences Building, in
what today might be considered minimal accommodations. Fortunately for
science, however, this close association brought them to collaborate on
a project that was concerned with the biosynthesis of sucrose, ordinary
table sugar. It was the bacteriologist Doudoroff who first observed the
phosphate-dependent cleavage of this disaccharide by the bacterium
Pseudomonas saccharophila, which produced glucose 1-phosphate and
fructose. The enzyme that catalyzed this reaction was purified and
named sucrose phosphorylase. Later, Doudoroff and Hassid, a
carbohydrate chemist, showed that the reaction could be reversed to form
a nonreducing sugar that appeared to be sucrose. At this time, Barker
joined the project, probably to bring his chemical training to bear in
proving the identity of the putative sucrose. When published, this
research led to an amusing incident, as described in Hassid 's obituary.
(8) "The enzymatic synthesis of sucrose resulted in some publicity that
came to the attention of officials of the Coca-Cola company, who were
having difficulty obtaining sucrose because of wartime rationing. The
company sent a representative to Berkeley to ascertain whether
commercial quantities of sucrose could be made by the enzymatic method.
Hassid and his associates were away on vacation at the time, so the
Coca-Cola emissary discussed the problem with (then Dean) Professor
Hoagland and reported that his company was prepared to provide $500,000
for research on this enzyme if a commercial process of sucrose synthesis
seemed feasible. Unfortunately, Professor Hoagland was pessimistic
about the possibility of sweetening Coca-Cola by this method, and so
further support of research on sucrose phosphorylase was left to the
University and the U.S. Public Health Service." Considering the
magnitude of this proposal, one can only wonder about the outcome had
Dean Hoagland left the matter for Barker, Doudoroff and Hassid to
negotiate with the Coca-Cola Company.

Barker has described his role in facilitating graduate study in
biochemistry at Berkeley by helping to start the Comparative
Biochemistry Group Major. In general, group majors are a device for
bypassing formalized departmental regulations in order to create
interdepartmental programs that accommodate to the special needs of
certain students and faculty. A perceived weakness, according to some,
is that the relaxed administration of such programs can lead to less
rigor in admission requirements and to poor supervision of the students.
Regardless, as Barker noted (3), "From 1936 to 1948 my students obtained
advanced degrees in the graduate curricula of Bacteriology,

xii

Microbiology, or Agricultural Chemistry. The Biochemistry Department at
Berkeley during that period was part of the Medical School; graduate
degrees in biochemistry were not available to students studying with
other faculty members. Since many students in other departments were
doing research on biochemical problems and wished to be recognized as
biochemists, there was considerable interest among both students and
faculty in setting up an academic mechanism for giving degrees in
biochemistry outside of the Biochemistry Department." Thus, in 1948,
Barker helped to establish a Ph.D. curriculum in comparative
biochemistry, which he administered until his retirement in 1975, and
during which time about seventy-five students were awarded degrees. As
he notes, the later performances of students such as Elizabeth Neufeld,
Paul Srere, and Earl Stadtman suggest that quality did not suffer during
his tenure in the program. In 1980, there were eleven such group majors
in the biological sciences, the one in comparative biochemistry
including faculty from biochemistry, cell physiology, chemical
biodynamics, chemistry, entomology, forestry, immunology, Lawrence
Berkeley Laboratory, molecular biology, nutritional science, physiology-
anatomy, public health, and UCSF biochemistry and biophysics. (9)
Clearly, the group majors have played a significant role on the Berkeley
campus .

In his formal teaching, Barker concentrated his efforts mainly in
two areas . One was an undergraduate laboratory course that he inherited
upon his appointment in 1937 from C. B. Lipman. This course initially
dealt with soil microbiology, but over the years it evolved into a plant
biochemistry laboratory. When Barker later joined the biochemistry
department in the College of Letters and Science, this course served as
a model for the Biochemistry 102L Laboratory that for many years was
offered by the department for nonmajor students. Barker also developed
a graduate lecture course in microbial metabolism in collaboration with
Doudoroff , and taught the course in the Department of Bacteriology with
Doudoroff, Roger Stanier, and Edward Adelberg. In all of his teaching,
Barker was straightforward, methodical, and well-organized, and he never
indulged in showmanship or intentional humor, nor did he consciously aim
to be entertaining. He gave his teaching the same serious consideration
he gave to his research, and my observations suggest that the students
respected him for this.

During his career at Berkeley, Barker appears to have been a
reluctant administrator, although he did chair the small departments of
plant nutrition (1949-50) and plant biochemistry (1950-53), and he
served two years as chairman of the biochemistry department (1962-64) at
the difficult time when it was preparing to move into new quarters and
sever connections with the Virus Laboratory. Years earlier, when the
Biochemistry and Virus Laboratory was completed in 1951, Barker had
moved onto the third floor along with several other members of the newly
formed Department of Agricultural Biochemistry. He has noted (3) that
"Although the laboratories were an improvement over those we had
previously occupied, the administrative arrangements in the building

X1X1

were difficult for several years because of an almost constant struggle
over authority and space." This is a muted reference to his
relationship with Wendell Stanley, who was recruited in 1948 to head up
the new biochemistry department in the College of Letters and Science
and to serve as director of the Virus Laboratory. (10) Fortunately for
all concerned, this source of conflict was eliminated in 1964 when most
of the biochemistry faculty moved to a new building at the west end of
the campus .

In one of the experiments carried out by Barker in Delft, he
observed the accumulation of large amounts of n-caproic acid during the
anaerobic fermentation of ethanol by a bacterium he had isolated by
enrichment culture, and Kluyver brought this result to the attention of
a local chemical manufacturer for possible exploitation. Barker
reports, however, "So far as I know nothing ever came of this.
Nevertheless, the company provided me with a small retainer that made it
possible, the following year, to start construction of a cabin in the
mountains of California we still use each summer." (3) This cabin,
built in 1937 at Silver Lake near Mount Lassen, is well-known to many of
Barker's friends and colleagues who have been lucky enough to spend a
few days there with him and his wife Margaret during the wonderful
summer days that visit the northern Sierras. They loved books and often
read to each other at home or at Silver Lake. The lake was also a place
for playing the word game Scrabble, and my wife and I often competed
with them in the evening before a roaring fire in their cabin. Margaret
always seemed to command the broadest and most esoteric vocabulary, but
her husband was a superior tactician when it came to utilizing the
available word combinations.

Visitors to the cabin were always taken on long hikes to the
numerous upper lakes in the so-called wilderness area, and along the way
they were introduced to every plant, insect, and mammal by the official
Latin names and were given a description of their most interesting
characteristics. On my first visit to Silver Lake about 1970, I asked
Barker if he had a map I could use while hiking by myself. He pulled a
sheet of yellow paper from a drawer and drew from memory all of the
trails, cliffs, ponds, and lakes for the surrounding five mile area, and
on it he indicated the best places to fish on each lake. My personal
map was done with such accuracy that I still use it today, some thirty
years later. The only thing it lacks is the location of the several
small "secret lakes" that Barker and his son Bob stocked early each
spring with fingerlings they caught and transplanted from the larger
surrounding lakes. After the fish matured for a year or two in these
food-rich lakes, the Barkers returned to harvest the reward. This and
other pleasant activities at the lake served to draw Barker back to his
cabin each summer for over sixty years.

After his retirement in 1975, mandated by the age limit then
enforced at the University of California, Barker maintained a regular
schedule of attendance on campus. Although he endured a heart attack

xiv

while hiking at Silver Lake in 1987 and had undergone bypass surgery, he
recovered quickly and his health remained good. For many of these later
years, he faithfully attended his wife, Margaret, during a prolonged
confinement that preceded her death in 1995. Shortly after his ninety-
third birthday, Barker suffered a brief illness and died from heart
failure at his home in Berkeley on December 24, 2000. (11)

In ending this review, it is fitting to recall the many honors
that accrued to Barker during his career (12), which include the Sugar
Research Award in 1945, election to the National Academy of Sciences in
1953, the Carl Neuberg Medal in 1959, the Borden Award in 1962, the
California Scientist of the Year Award in 1965, the F. G. Hopkins Medal
of the British Biochemical Society in 1967, the National Medal of
Science in 1968 presented to him at the White House by President Lyndon
Johnson, and the University of California Berkeley Citation in 1975. In
1988, the Biochemistry Building was renamed Barker Hall and Barker's
portrait was hung in the lobby where it will long bring enduring
recognition to this remarkable man.

Clinton E. Ballou, Ph.D.
Professor Emeritus, Biochemistry

February, 2001

University of California, Berkeley

References

1. Barker's son, Bob, suggested (December 24, 2000) that "Nook" is
derived from "Snookums." According to The World Encyclopedia of
Comics (Maurice Horn, ed., Chelsea House Publishers, 1976),
Snookums is the name of the infant character in the comic strip
"The Newlyweds" by George McManus, published as a newspaper Sunday
feature from 1904-1918.

2. Personal comment by H. A. Barker.

3. H. A. Barker, "Explorations of Bacterial Metabolism," Annual
Reviews of Biochemistry 1978, 47:1-33.

4. H. A. Barker curriculum vitae and bibliography (1978?).

5. H. A. Barker, "Notes on the history of biochemistry at Berkeley,"
December 15, 1969.

6. J. R. Quayle, personal communication, January 26, 2001.

7. H. Weissbach, personal communication, January 5, 2001.

8. C. E. Ballou and H. A. Barker, "Willaim Zev Hassid (1899-1974) A
Biographical Memoir," Proceedings of the National Academy of
Sciences 1979, 50:197-230.

9. University of California, Berkeley, General Catalog 1980.

10. A. N. H. Creager, "Wendell Stanley's Dream of a Free-standing
Biochemistry Department at the University of California,
Berkeley," Journal of the History of Biology 1996, 29:331-360.

11. H. A. Barker obituary, San Francisco Chronicle, January 5, 2001.

12. American Men and Women of Science, 15th edition, R. R. Bowker Co.,
1982.

xvi
INTERVIEW HISTORY—Horace A. Barker

Horace Barker was interviewed for the Bancroft Library's Program
in the History of the Biosciences and Biotechnology as part of its
effort to document basic science contributions to biomedicine and the
biotechnology industry. From the 1930s on, Barker pursued a basic
biochemical approach to microbiology focused on natural history and
metabolism of soil bacteria at a time when many others in the field were
studying microorganisms as pathogens.

We are grateful to Dr. Barker for persevering at age ninety-one
and despite ill health through three interview sessions in which he
provided the outline of his professional achievements, particularly
those early in his career. Highlights of his story are his two summers
and one fellowship year in the 1930s with the eminent Dutch
microbiologist, Cornelius van Niel at Stanford's Hopkins Marine Station;
his postdoctoral fellowship under A. J. Kluyver at the Delft Laboratory
of Microbiology in Holland, and his long service, 1936-1975, on the
Berkeley faculty, first in the University of California Agricultural
Experiment Station and later in the department of biochemistry.

Of particular interest is Barker's work beginning in the late
1930s with the use of artificial radioisotopes produced by Ernest
Lawrence's 60-inch cyclotron in Crocker Radiation Laboratory on the
Berkeley campus. Barker tells in the oral history of his collaboration
with the physical scientists Martin Kamen, Sam Ruben, and Zev Hassid in
some of the earliest work anywhere using artificial radioisotopes in
biological tracer experiments. His account is a useful extension of the
series of oral histories in the Bancroft Library on medical physics at
Berkeley, which include documentation of the earliest synthesis and
application of artificial radioisotopes in biology.

We would like to have heard more about Barker's many other
accomplishments, unfortunately only partially recounted here, in
instilling comparative microbial biochemistry at UC Berkeley. His
renown rests on "a lifetime record of stellar achievements" in basic
science, as the Nobel laureate Arthur Kornberg commented.1 Barker
performed pioneering work on elucidating metabolic pathways in soil
bacteria, including his work on vitamin B12 for which UC held a patent
and which became of considerable interest to the pharmaceutical firms
Merck and Squibb. Thus Barker's work presents an early example of the
commercial potential of basic biological research well before the
recombinant DNA revolution of the 1970s and the growth of the modern

1 Arthur Kornberg, For the Love of Enzymes: The Odyssey of a Biochemist,
Cambridge: Harvard University press, 1989, p. 172.

xvii

biotechnology industry. Fortunately, Barker some years ago carefully
arranged and then donated to the Bancroft Library more than eleven
cartons of his correspondence, laboratory notebooks, and assorted
photographs documenting his scientific contributions. His lengthy
review of his own scientific career, published in the Annual Review of
Biochemistry, is available in the appendix of this oral history, along
with his curriculum vitae, bibliography, and other relevant documents.
These documents help to fill in what Dr. Barker left out of the oral
history, but fail to provide the social context and personal dimensions
of his activities.

Also missing in the oral history is documentation of Barker's role
in helping to recruit many of the biochemists and molecular biologists
who were to make UC Berkeley a center of the biochemical and molecular
approach in the life sciences. Barker does however mention the Program
in Comparative Biochemistry, an interdepartmental group which provided a
broad forum for faculty and graduate students interested in
biochemistry. He also hints at, but falls short of elaborating on, the
animosity between Wendell Stanley and his group in the Virus Lab, and
the biochemists whom Stanley had hoped to unite in Berkeley's first
department of biochemistry, founded in 1950. Barker was in fact one of
Stanley's prime opponents, objecting among other things to Stanley's
attempt to focus biochemistry on viral research. As a result of these
and other professional and personal tensions, Stanley resigned in 1953
as chairman of biochemistry. In the end, three separate departments
emerged from the group that Stanley had striven unsuccessfully to unite:
biochemistry, virology, and molecular biology. Thus Stanley's vision of
a unified biochemical and molecular enterprise on campus was only
realized in the 1980s and nineties when these fields were organized
under a new Department of Molecular and Cell Biology. For full
historical treatment of these developments, the reader is referred to an
article by Angela Creager2 and an oral history in The Bancroft Library
series on the reorganization of biology at Berkeley.

Oral History Process

Three interviews were conducted with Dr. Barker between December
21, 1998, and January 14, 1999. The first was conducted in Dr. Barker's
office in Barker Hall on the Berkeley campus, with biochemistry
colleagues Clinton Ballou and Edward Penhoet in attendance. We are
grateful to Dr. Penhoet for instigating the idea for and funding
interviews with his mentor Dr. Barker. The later interviews were
conducted one to one, in Barker's modest home in Berkeley where he lived
alone after the death of his wife Margaret in 1995. Soft spoken and

2Angela N. H. Creager, "Wendell Stanley's Dream of a Free-standing
Biochemistry Department at the University of California, Berkeley," Journal of
the History of Biology 1996, 29:331-360.

xviii

reserved, Dr. Barker answered to the best of his ability but had trouble
remembering details of recent history. We thank Dr. Barker's daughter,
Barbara Friede, and Louise Taylor, long a friend of the Barker family,
for reviewing the transcripts. They made only very occasional changes
and additions.

We are particularly indebted to Clinton E. Ballou, Ph.D., for
extending the information and accuracy of the oral history. He
painstakingly reviewed the transcripts, provided biographical
information for people mentioned by Dr. Barker, corrected spelling of
proper names, and so on. In doing so, Dr. Ballou pulled on his long
association with Barker as colleague and friend in the Berkeley
biochemistry department. As emeritus professors, they shared an office
in Barker Hall, the location of the first interview. In addition, Dr.
Ballou carefully researched and wrote the introduction to this volume.
Although there is no equivalent to an oral history of Dr. Barker
recorded in his prime, thanks to Dr. Ballou 's contributions, for which
we are truly grateful, the present volume is the next best thing. Dr.
Ballou 's introduction describes Barker's scientific contributions and
fills in details which Dr. Barker was unable to provide about the
postwar development of biochemistry on the Berkeley campus. As a
result, we believe that by using the combined resources of the
introductory material, the interviews, and the appendix contents, the
reader will obtain a good sense of Dr. Barker and his science. For the
serious researcher, this oral history will provide a useful platform for
further research.

This oral history reflects the contributions and working
environment of a remarkable scientist who is widely respected and did
much to advance the field of comparative microbiology, particularly in
the area of bacterial metabolism.

Dr. Barker died quietly at home on December 28, 2000, before the
oral history volume was completed.

The Regional Oral History Office was established in 1954 to
augment through tape-recorded memoirs the Library's materials on the
history of California and the West. Copies of all interviews are
available for research use in The Bancroft Library and in the UCLA
Department of Special Collections. The office is under the direction of
Richard Candida Smith, Director, and the administrative direction of
Charles B. Faulhaber, James D. Hart Director of The Bancroft Library,
University of California, Berkeley.

Sally Smith Hughes, Ph.D,

Historian of Science and Project Director

July 2001

Regional Oral History Office

The Bancroft Library

University of California, Berkeley

xix

Regional Oral History Office University of California

Room 486 The Bancroft Library Berkeley, California 94720

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INTERVIEW WITH HORACE BARKER

I HIGHER EDUCATION, 1926-1936
[Interview 1: December 21, 1998] : ##2

Stanford University, 1926-1933

Research Assistant with C.V. Taylor, 1930-1931

Hughes: Well, do you want to start with your undergraduate years at

Stanford [1925-1929]? I know from your review article3 that you
had a hard time deciding whether it was going to be the
humanities or the sciences that you were going to major in.

Barker: Well, my family- -my brother in particular- -had been in the
humanities. He ended up as an English professor. He was a
Rhodes scholar and he was at Oxford for several years where I
visited him briefly. He was at Stanford after that but then he
ended up in the eastern United States for most of his career.

Hughes: Do you remember how you decided on the sciences rather than the
humanities?

Barker: Well, I got started in biology; that's really why I got into
science. When I was an undergraduate I got invited by a
professor in the biology department, whose name escapes me at the

moment .

Hughes: Taylor?

1 Also present: Edward E. Penhoet and Clinton E. Ballou.

2 ## This symbol indicates that a tape or tape segment has begun or
ended. A guide to the tapes follows the transcript.

3 H. A. Barker. "Explorations of bacterial metabolism." Annual Review
of Biochemistry 1978, 47:1-33.

Barker:

Hughes:
Barker:

Hughes :
Barker:

Hughes :

Barker:

C. V. Taylor. I worked with him somewhat and then he was invited
to the University of Chicago and invited me to come along as a
research assistant, so I was there for a year [1930-31].

Was it unusual for an undergraduate to go off with a professor?

Let's see, was I an undergraduate at the time? I got my A.B. in
1929, I believe.

Yes, you're right.

So I was a young graduate student. January term I took a course
from Taylor. And it was a very small course; I think there were
only about half-a-dozen students in it. Evidently I clicked in
some way with him and so he invited me to come to Chicago. So I
learned about the stockyards. [laughs]

Did that experience get you interested in microbes?
remember, was a protozoologist.

Taylor, as I

Yes, he was a protozoologist. Well, the thing that really got me
started in microbiology was a course I took with [Cornelius] van
Niel one summer at Pacific Grove [California] . Van Niel was a
very prominent man in the area of microbiology. He discovered
most synthetic bacteria and a variety of other things. He left
Holland shortly after he got his Ph.D. and came to Pacific Grove,
and I was one of his first students.

Thesis in the Chemistry Department

Penhoet: Did you do your thesis work at Pacific Grove?

Barker: No, I did my thesis in chemistry at Stanford, with [James W. ]
McBain.

Hughes: And what was the subject?

Barker: Well, let's see, it was a long time ago.

Hughes: Was it microbial?

Barker: No, I worked on egg albumen- -denaturation of egg albumen.

Penhoet: Oh, so it was biochemistry.

Barker: It was biochemistry.

Penhoet: In the chemistry department at Stanford. That's interesting.

Barker: Well, see, [Murray] Luck was a biochemist. He was in the

chemistry department for many years. A He wasn't a very good
teacher; his main activities were in the Annual Review [of
Biochemistry] , I think.

Hughes: So it sounds as though from a relatively early age you were

interested in the biochemical aspects of microorganisms, is that
right?

Barker: Right.

Hughes : How did you get on that track?

Barker: Well, I think it was probably initially from my contacts with
C. V. Taylor. He was later the chairman of the biology
department at Stanford for a number of years.

Hughes: But he had a biochemical interest?

Barker: Well, he had a biochemical interest, yes, but he was really more
of a biologist than he was biochemist.

Penhoet: Going further back, Nook, undergraduates at Stanford were
encouraged to get involved in research? You told me, for
example, that several summers you took a car with another
undergraduate and drove all over the southwest collecting plants,

Early Interest in Botany

Barker: But that was unconnected with any push by anybody on the faculty.
At Stanford in the early days I got interested in botany. I took
a course in systematic botany with [L. R. ] Abrams, who was in
charge of the herbarium at Stanford. It was a course in which
you took field trips and went out to various places around the
Bay Area. And once we went up to Yosemite, I remember.

We collected various things and brought them back and
classified them. It was a rather traditional taxonomy course.

* Luck was a biochemist in the Department of Chemistry; in 1959, Stanford
created its first department of biochemistry, with Arthur Kornberg as
chairman. For a history of the latter department, see the oral history with
Kornberg in The Bancroft Library Series.

Penhoet:
Barker:

Hughes:
Barker:

Penhoet:

Barker:
Ballou:

Barker:

Hughes:
Barker:

But I got interested since I had been going into the mountains
for many years and I began collecting things on my own. I had a
substantial collection which I contributed, ultimately, to the
herbarium of Stanford. I don't know what's happened to it since
then, but it was moved to San Francisco at one stage, to the
Academy of Sciences.

So your interest in nature came from your own family experience.

Well, it came from the mountains, really. Yes, at some stage I
started collecting plants in the mountains.

This was before you were even an undergraduate—when you were a
child?

Oh, well, I'm sure to some degree it had an influence, but I
didn't really begin collecting in any formal way until I was a
student .

This was something that you did on your own.
connection with any class.

It wasn't in

Well, I took a course in systematic botany at Stanford and I
think that stimulated it. We took a number of field trips and we
went to Yosemite in the fall once. We went to various places to
collect so later on I took this more seriously than I did on my
own. [laughs]

Was that typical of the education at Stanford at the time, that
students got involved in field trips and doing things, rather
than just reading out of books?

Oh, yes, I think so. At least as they began to get into some
research area, or potential research area.

How many students were there at Stanford at that time, do you
have a recollection? It must not have been a very large
university, was it?

No, it wasn't really large. I don't know how many, but it was
much, much smaller than Berkeley in those days.

There was a lot of contact between students and faculty?
Yes. There was quite a lot of contact with faculty.

Cornelius van Niel and the Hopkins Marine Station. Pacific Grove.
California. 1931-1935

Van Niel as a Personality

Hughes: Well, let's go back to van Niel and the Hopkins Marine Station.

Barker: All right.

Hughes: Could you say something about him as a personality?

Barker: Oh, he was a very impressive guy. [laughs] He was very, very
outgoing, and he had an impressive way of speaking.

Hughes: Was he fluent in English?

Barker: Very fluent in English, oh, yes. In Holland, you know, they
learn German, French and English, in grammar school, I guess.
Yes, he spoke quite fluently. He had a slight Dutch accent,
[laughs] And he was a great teacher.

Hughes: What was his approach to microbiology?

Barker: Well, he had a great interest in the biochemical aspects.

Hughes: You were there [at Hopkins Marine Station] two summers [1930,

1931], and then also for a National Research Council Fellowship
[1933-35] .

Barker: Yes, I was.

Work with J. P. Baumberger, Summer 1931

Hughes: Did van Niel have projects for you to do, or did you come with
projects in mind?

Barker: Well, let's see, I think the first time 1 was there was not with
him; it was connected with a professor whose name escapes me at
the moment, who was in the physiology department at Stanford. Do
you have any names there?

Hughes: Yes, there's a J. [James] P. [Percy] Baumberger.

Barker: Yes, Baumberger. I think I was originally an assistant in some
of his classes. I didn't get acquainted with van Niel until
later.

Hughes: Not until your fellowship?
Barker: Yes.

Hughes: How did a day go? Were you working shoulder to shoulder, so to
speak, or did everybody have more or less independent projects?

Barker: Well, the Hopkins Marine Station is a very small place, and so at
that time there were two sets of activity. There were some
people who were interested in invertebrates . They would get up
early in the morning and collect when the tide was low, that sort
of thing. I did a little of this because we did it in connection
with classes.

Working with van Niel

Barker: But then van Niel came and he had no direct connection, I think,
to this sort of thing. He had been trained in Delft in Holland
by A. J. Kluyver, which is sort of a famous name in general
microbiology. Van Niel had a very impressive personality, as a
teacher and person. [laughs] He had a manner of talking that
really was highly impressive. He had a big effect on people, I
think, in personality.

Penhoet: The summers that you went to work in his lab, were there a lot of
people, or just a few?

Barker: Oh, just a few.
Penhoet: A handful.

Barker: Yes, I was perhaps his first student at that stage. I began
working with him. In those days, one had very close contacts
with the professors.

Penhoet: So you really worked with him all day, every day, when you were
there for the summers?

Barker: Yes. Sometimes we did.

Van Niel's Microbiology Course

Hughes: Was his course hands-on?

Barker: Oh, yes. Well, I think he was probably the most famous teacher
in microbiology of his age.

Hughes: Famous people have taken his course.

Barker: I was one of his first students. He used to give me lectures on
alcoholic fermentation, for example. He would spend three hours
[laughs] lecturing me, and without any notes. I know he studied
very hard at night to prepare his lectures.

Ballou: Just for you?

Barker: Well, just for me, to begin with, yes. [laughter]

Penhoet: So he gave you a three-hour lecture just by talking one on one?

Barker: Yes, that's right.

Penhoet: Oh, fantastic!

Hughes: My understanding is that there was never more than a handful of
students for the course each summer.

Barker: Well, he was never able to handle more than perhaps ten at the

outside, I would guess. But he got people who later became quite
well known.

Hughes: My understanding, too, is that it wasn't just the microorganisms
themselves, it was also a whole technology that he was teaching,
enrichment culture, for example.

Barker: Yes.

Hughes: How widely known was enrichment culture?

Barker: Well, that was a technique which was developed mainly in Holland,
well, mainly by [Martinus] Beijerinck. He was the original
microbiologist at the University of Delft, starting maybe in the
1890s sometime. And he lived until sometime in the early
twenties.5 I think van Niel actually took some courses from him.
But the one van Niel was most closely connected with

Beijerinck died in 1931.

intellectually and spiritually was A. J. Kluyver. Van Niel was
his successor.

Hughes: Wasn't Kluyver a student of Beijerinck1 s?

Barker: Yes.

Hughes: And van Niel was a student of Kluyver.

Rockefeller Foundation Fellow, Microbiology Laboratory,
Polvtechnical School6. Delft. 1935-1936

Physical Layout and Operation of the Microbiology Laboratory

Barker: All this induced me after I got my Ph.D. to spend a year in
Holland.

Hughes: Tell me about that, because the Delft school is famous.

Barker: Kluyver lived in a big house on the canal, and this house was
directly connected with the laboratory.

Hughes: And Beijerinck had lived there before him?

Barker: I think so, yes. The apartment was right on the canal. You

could look out the window and see the canal boats sailing past.

Hughes: What were the laboratories like? How were they equipped?

Barker: Oh, they were very well equipped for those days. And there were
various assistants—people that were paid to take care of
specialized equipment, to keep track of things and so on. They
had a very big collection of microorganisms dating from
Beijerinck's time.

Hughes: So they had a series of cultures that were being maintained?

Barker: Oh, yes. Kluyver always had an assistant. It was really a full-
time job for somebody who had usually gotten a Ph.D. in the lab
or was getting a Ph.D. in the lab. Van Niel had been this at one

' The name in Dutch is Technische Hoogeschool. The literal English
translation "technical high school" more correctly translates as polytechnical
school or college.

time, and when I was there it was a man by the name of Holdgers
who later on became a professor of microbiology in Amsterdam.

Hughes: Once again, this was a metabolic approach, was is not?

Barker: Yes. There was no medical application involved.

Hughes: Was the Delft school a technical school?

Barker: Yes.

Hughes: What difference did that make to the type of research that went
on there?

Barker: Well, I don't know that it made any difference, because I had
virtually no contacts [with the school]. Except, I remember I
was interested in learning something about micro-chemistry-
chemical tests on small amounts of things. And Kluyver arranged
for me to take sort of a private course with a professor in the
chemistry department there, in techniques.

Hughes: At the university?

Barker: At the university there.

Hughes: How big a group was it at the Delft school?

Barker: Oh, well, let's see, I don't know about the university in

general, because I had virtually no contact with it, but the
microbiology lab had maybe six or eight or ten people doing
Ph.D.s. And then Kluyver also taught a course--! think it was
two lectures a week—for undergraduate students. That was the
only time we saw undergraduates.

Hughes: So it was considered the department of microbiology for the
university?

Barker: Yes. It was part of the university.
Hughes: Were you unusual in being a foreign student?

Barker: Well, they had had a succession of foreign students there who had
become quite well known.

Hughes: Had you already learned enrichment culture?
Barker: Yes, from van Niel.

Research on Methane-producing Bacteria

Hughes: What did you do that year?

Barker: Well, I became interested in methane-producing bacteria. Holland
was a good place to study them because the canal is there,
[laughs] And [other] wet places.

Hughes: Is that one reason you chose Delft?

Barker: Oh, no, I went there because van Niel had been there and

recommended Kluyver and so on as being a very fine place to be
for microbiology.

Hughes: Well, why study methane bacteria particularly?

Barker: Well, I got interested in methane bacteria at Pacific Grove,

before I went to Holland. We used to go out occasionally with
van Niel on collecting trips around the Monterey area and bring
back samples of mud from this, that, and the other place and set
up enrichment cultures—cultures in which you'd get methane
bacteria. You need an anaerobic environment, so you had some
sort of container or bottle which you filled more or less
completely with some mud in the bottom, and then some normal
nutrients for living organisms. You really want organic
compounds --things like acetate or ethyl alcohol, succinic acid--
almost anything of that sort, a single compound. You let it sit
awhile; pretty soon it begins to bubble, and bubble, and bubble,
and it gets more active and pretty soon you have a pretty good
enrichment culture.

Ballou: So how do you determine that you have methane-producing bacteria?
Barker: Well, you'd collect some of the gas that's coming off.
Ballou: And light it with a match? [laughs]

Barker: And do a gas analysis on that. We had some sort of system in
Pacific Grove for analyzing gas.

Penhoet: So even then it was a little bit of an outgrowth of your interest
in collecting things in nature.

Barker: Oh, yes.

Penhoet: But now moving into biochemical analysis of these things in
nature.

Barker: Right. Before this I collected plants,

The Enrichment Culture Technique

Hughes: Would it be obvious how one should enrich the culture when you
were trying to favor one culture over another?

Barker: Beijerinck was the one who originated the enrichment culture

technique. He pushed it very hard. So you'd take a compound and
put it in a particular environment. If you wanted an anaerobe,
you filled up the bottle with mud and put in the compound and
then you waited until something happened. And pretty soon things
would begin bubbling, and you knew you had an organism that used
this compound and that it probably wasn't isolated, and you could
find out more specifically what it did. In many cases it would
be a combination of organisms. For example, if you had alcohol
as a substrate, it usually would get oxidized to acetate, and
then methane bacteria would work on the acetate. There's usually
some mud in the bottom, and you'd shake the culture a little and
a lot of bubbles would come out.

II BIOCHEMIST AT BERKELEY, 1936-1975

Faculty Member and Soil Microbiologist, UC Berkeley Agriculture
Experiment Station. 1936-19507

Penhoet: We were talking about the medical school being separate from the
rest of the activities on the [Berkeley] campus.

Barker: Well, originally biochemistry was part of the medical school, and
bacteriology also, I believe, under Karl [Friedrich] Meyer.

Hughes: But biochemistry was also located here?

Barker: In Life Sciences [Building]. Biochemistry was on the ground

floor and bacteriology was on the third or fourth floor, I think
it was at the time.

First Position at Berkeley

Penhoet :

Barker:

How is it that you were in the plant group8 although your work
had always been with microbial systems?

Well, I was in Holland when I got an invitation to come here,
[pause] Well, I'm not really quite sure why they invited me.
suppose they had an opening at that time.

7 A portion from the third interview session is included in this section.

8 From 1936 to 1969, Dr. Barker was on the faculty of the Agricultural
Experiment Station, UC Berkeley. In 1949-50, he was chairman of Berkeley's
Department of Plant Nutrition. For subsequent appointments and departmental
name changes, see his "Biography for Academic Personnel" at the back of this
volume .

Penhoet: So it was just an accident of history that the plant group had an
opening?

Barker: Well, I don't know whether it was an accident or there was

something of more rationality in it, but at any rate I'd never
really had any connections with agriculture before. At Stanford
I was in the chemistry department, and then I was in Pacific
Grove with van Niel for a time. And then I was in Holland. But
when I came [to Berkeley], for a number of years the course I
taught was soil microbiology.

Penhoet: I see. So that was the connection to agriculture.

Barker: Yes, all the agriculture students at that time had to take a
course in microbiology. And I became the one who taught them
there in the lab of the Life Sciences Building.

Hughes: Well, that makes sense, with your background, that you would be
invited to teach soil microbiology. You were appointed Junior
Soil Microbiologist as well as Instructor [1936-1940].

Early Faculty Members in the Berkeley Agriculture Program

Barker: I received an invitation to come to Berkeley I suppose through

van Niel's influence. Professor [Dennis R. ] Hoagland was looking
for somebody to teach soil microbiology to the students of
agriculture here.

Hughes: Tell me about Hoagland.

Barker: Hoagland was a plant physiologist, well known here. He was
interested primarily in nutrition of higher plants.

Hughes: Was Hoagland doing mainly applied research?

Barker: Well, he did some applied research, but he was interested

basically in finding out what elements were required for the
growth of plants—usually in water culture—and then seeing what
quantities were involved and that sort of thing.

Hughes: Were you the only soil microbiologist?

Barker: Well, before me the person who had taught soil microbiology for a
number of years was C. B. Lipman, who was dean of the graduate
school. He had a great interest in soil microbiology, so he'd
come over two or three afternoons a week and teach a course in a

rather informal way and then go back to his more official office
and carry out [laughs] the higher activities of the university.
Yes, he was quite a character.

Hughes: Why do you say that?

Barker: Well, I think that Professor Hoagland had a very broad view of

research and what its implications were and what it was good for
and so on. He was quite supportive. After all, a lot of the
bacteriology I did was not immediately related to agriculture.

Hughes: Do you think he realized that when he appointed you?

Barker: Well, he appointed me because he got in touch with van Niel, with
whom I had worked as a postdoctoral fellow. And then he wrote to
me when I was still in Holland and offered me this position in
soil microbiology. I don't know whether Dean Lipman had
something to do with it, too.

Hughes: If Hoagland hired a faculty member who was going to carry the
soil microbiology course, then that must have been enough to
justify your position in the College of Agriculture. My point is
that even though your research might not have had direct
agriculture applications, you were certainly helping out the
agricultural curriculum by teaching the soil microbiology class.

In those days did heads of departments or divisions have
more freedom in whom they hired and the sort of research that was
done? Was there less need for justification?

Barker: Oh, I don't know how they justified hiring me. [laughter] They
just 'did. There were a number of universities in the United
States where soil microbiology was given considerable support.
Wisconsin, for example, was one, and Illinois, they all had
courses that had been going for quite a long time and were quite
successful.

Hughes: Was Lipman the first to teach it here?

Barker: Yes, I think he was probably the first one who taught soil

microbiology on the campus. He enjoyed doing some sorts of soil
microbiology himself. Although he was dean of the graduate
school and had been I think for a number of years and continued
throughout the early years when I was here, he usually would come
to his laboratory in the Life Sciences Building in the morning
and spend an hour or two there and then would go to the dean's
office and do what he had to do there. He did that for a number
of years certainly, but then when soil microbiology got started
as a formal course, it was really too much work for him to

Hughes:

handle—to give all the lectures and take care of the laboratory
and so on- -and so they got me to help out.

Well, you were at the Agriculture Experiment Station, which I
presume was located in LSB. Am I right?

Barker: I was always in the Life Sciences [Building] .9

Hughes: Yes, but according to the records, technically your appointment
was in the Agriculture Experiment Station, UC Berkeley.

Barker: Yes, that's right. Well, the Agriculture Experiment Station was
a big affair. It covered a wide area of agriculture—Davis and
Berkeley.

Penhoet: So it was an administrative structure?

Hughes: It was an administrative structure, yes.

Ballou: Well, where was it located on the Berkeley campus?

Barker: The dean had an office in one of these agriculture buildings, he
and the assistant dean. But it was a big activity. You know, in
addition to people who were teachers and professorial types, they
had people who didn't have a very direct connection with the
university but contact with agriculture people throughout the
state.

Hughes: It still exists at the state level.
Barker: Yes, I'm sure it still exists. [laughter]

Hughes: The plant nutrition group10--which was your group, am I not
right—was in LSB?

Barker: That's it.

Hughes: And then were there other groups in the Agriculture Experiment
Station elsewhere on campus?

9 Dr. Barker later moved to Stanley Hall.

10 The academic units with which Dr. Barker was affiliated underwent
numerous name and organizational changes, too complicated to outline here,
See his curriculum vitae at the back of this volume.

Barker: Oh, yes, in Agriculture Hall and Giannini Hall. There was food

technology, for example, where [Emil M. ] Mrak got his start. You
know about Mrak?

Hughes: No.

Barker: Well, he was [chancellor] at [UC] Davis for a number of years.

Hughes: How did the conditions at LSB compare with your past experiences
at Delft and the Hopkins Marine Station?

Barker: Well, initially the lab was a big lab with nothing much in it but
a warm room and a place to sterilize glassware. There wasn't
much else.

Penhoet: Who supplied the money for your research in those days?

Hughes: The Agriculture Experiment Station. I don't remember how much I
got, but it was enough to get a lab started and then money was
available. You see, my appointments were academic and in the
experiment station. The experiment station had the money and so
on and supplied what was needed.

Hughes: That would have been State of California money?

Barker: I think so. Then I also had a grant from the National Science
Foundation.

Teaching

Early Courses

Hughes: Were you teaching right from the start?

Barker: Yes. I'd really never done any teaching to speak of, except for
helping at van Niel's course at Pacific Grove. When I came, I
didn't have the course all to myself. C. B. Lipman was in charge
of the course for the first year or two, and then I sort of took
charge of the laboratory work.

Hughes: And this was soil microbiology.

Barker: It was soil microbiology, in which initially there was one

lecture a week which Lipman gave very informally. I usually

attended that and sort of got an idea of what he was talking
about.

Hughes: What was your part in the course?

Barker: Well, initially I just went to listen to him, but I was in charge
pretty much, under his general direction, of the laboratory. He
was pretty busy, so sometimes he'd drop in a lab but generally
that was pretty much my area.

Ballou: What kinds of experiments did you do in the teaching lab?

Barker: Well, we had a syllabus of some sort, which I worked up.

Initially Lipman didn't have anything; he sort of played it by
ear. [laughter]

Ballou: You would isolate organisms?

Barker: Yes. We would isolate sulfur-producing bacteria, and this, that,
and the other thing.

Penhoet: How many students were there?
Barker: Well, initially there were two.
Penhoet: Two students?

Barker: Two students. [laughs] In Lipman 's days classes were very

small. Shortly after I came it became a required course and then
we had about eighteen or twenty.

Ballou: A good way to increase enrollment is to make the course required
for some major. [laughter]

Barker: Yes, well, all the students in the soils curriculum were required
to take this course.

Hughes: What were their career aspirations?

Barker: Well, there were a variety of [positions] in California for

people [to give] advice in the agriculture community. I think
many of them went into that sort of thing. Some, of course, got
out of agriculture entirely and got into other areas-
microbiology. One of my best known students was Earl Stadtman,
who was at the National Institutes of Health for quite a number
of years.

Hughes: He did research with you as well?

Barker: Yes.

Hughes: I noticed his name on some of your papers.

Barker: Well, part of it was Ph.D. work.

Hughes: Let me go back to the teaching for just a minute. You had a very
basic approach to microbiology; you were interested in the
biochemistry of these organisms.

Barker: Yes.

Hughes: Did you have to change that focus when you began to teach soil
microbiology?

Barker: It wasn't so terribly different from what I'd learned from van
Niel. Well, there was appreciably more application to
agriculture, per se. The organisms might be of some importance
to the growth of plants—nitrogen fixation, nitrification, and
things like this .

Hughes: Did you teach a metabolically oriented course?

Barker: Well, to some degree, but the students that we had in agriculture
didn't have very extensive backgrounds in biochemistry.

The Program in Comparative Biochemistry

Barker: Later on, I developed a course with several members of the

bacteriology department that was intended to teach bacterial
metabolism.

Hughes: Is that the Program in Comparative Biochemistry?
Barker: Yes. I was the chief administrator in biochemistry.
Hughes : Why did you help to set up the program?

Barker: Well, we needed it for students. One or another of the [faculty]
within the program had gotten many of their students [through
this program] .

Ballou: It's really a mechanism for going across departments to attract
students .

Barker:

Yes, it is.

Ballou: But it existed for more than ten years.
Hughes: Did it?

Barker: Oh, a long time. In fact, up until the reorganization of biology
occurred here [in the 1980s and early 1990s].

Penhoet: But there was no biochemistry in any specific place [before
1950].

Barker: There was biochemistry but it was in the medical school [at UC

San Francisco]. [David] Greenberg was chairman of the group, and
there were a few other people in it.

Ballou: Right. Was it Herbert Evans whom you were thinking about in
biochemistry?

Barker: No. Herbert Evans had no contacts really at all with us. He was
a unit unto himself. I don't think he encouraged or had much
contact, although I knew a few people who worked with him.

Hughes: Who was involved in the Program in Comparative Biochemistry?

Barker: Well, it was mainly bacteriology and agriculture.

Penhoet: Was Mike Doudoroff one of the founders of the program with you?

Barker: Yes, he for several years served as chairman of the microbiology
group and the bacteriology department.

Schism in the Department of Bacteriology

Barker: The bacteriology department was strongly divided between medical
and nonmedical. They didn't really talk to each other at all, to
speak of! [laughter] And they taught courses that were quite
unrelated.

Hughes: [Israel Lyon] Chaikoff is a name-
Barker: He was in physiology.

Hughes: He, too, was interested in getting the Program in Comparative
Biochemistry off the ground?

Barker: Yes, he was one of the people who had students in comparative
biochemistry.

Hughes: But he was medically oriented, was he not?

Barker: Well, he was in physiology.

Hughes: But you talked to him. [laughs]

Barker: Oh, yes. [laughs]

Penhoet: Well, I think he was talking about the schism in the bacteriology
department. It was part medical people and part others, and they
are the ones who didn't talk to each other.

Barker: No, there was scarcely any communication between the medical and
the nonmedical bacteriologists. It was understandable; they
dealt with entirely different organisms.

Hughes: Was that characteristic of bacteriology departments of that era?
Barker: I think so, yes.
Hughes: There was a schism.

Barker: Yes, in places like Wisconsin and so on they had a general

bacteriology department as well as a medical school bacteriology
department. [laughs] I think it was terribly common.

Research with Radioactive Tracers11

Collaborations with Sam Ruben and Martin Kamen

Hughes: Let's go back to your research using some of the early

radioactive tracers. I understand that you worked with Kamen.

Barker: Martin Kamen.

Hughes: Actually, that was later, wasn't it?

Barker: Sam Ruben was the initial person.

Hughes: With carbon-11, right?

11 A discussion of tracers from the third interview session is
incorporated here.

Barker: Yes.

Hughes: How did that relationship begin?

Barker: Well, [Zev] Hassid was a friend of Sam Ruben, and it was through
Hassid that I got connected with Ruben. We often spent most of
the night waiting for the cyclotron so we could prepare some C1A,
and then [spent] the rest of the night [laughs] getting it in
shape so we could use it. And so I guess for a time I was the
only one outside--at least outside the chemistry department- -that
used Cu.

The people in the chemistry department had this nice tool
that could be used and didn't really know how to use it.
[laughs] They had people that I knew didn't have any background
in physiology and so on so they thought it was a godsend to have
microorganisms that could do all sorts of things. You didn't
have to bother with patients and so on. All you had to do was
take a little mud or something of the sort for a culture to do an
experiment .

Penhoet: Where did the use of radioactivity as a tracer in determining
biochemical patterns start? It started here?12

Barker: It started here. Yes, I think so. Let's see, there were some
people who used heavy isotopes in other places, but as far as
radioactive isotopes are concerned, it really started here.

Ballou: Martin Kamen wrote a very nice summary of this published in

Science a number of years ago. He analyzed very precisely just
where the first experiment was done and where the ideas came
from.

Penhoet : So you were a natural to work on this .

Barker: [slowly] Well, yes. The two senior people were Ruben and Kamen.
Kamen was the physicist; Ruben was the chemist. Initially,
Kamen1 s job was just to prepare the isotope, to go through the
procedures which are necessary.

Hughes: How did you detect the radioactive compounds?

Barker: Well, Ruben had counters, and later on I think I had a counter,
too, that I used in the Life Sciences Building. But initially
Ruben and Martin Kamen were the two people who were responsible

12 For more on the origins of the use of artificial radioisotopes , see
the Bancroft Library oral history series on medical physics at Berkeley.

for the physical aspects of the preparation and counting and so
on.

Ruben was an instructor, I think, at that time and maybe an
assistant professor in the chemistry department. And Martin
Kamen was in the Radiation Lab, but they used to collaborate.
Kamen was generally involved simply in the preparation of the
Cu, and Ruben and I were involved in doing the experiments.
Kamen would come past and watch in the early hours of the morning
[laughs] and see if anything would happen that was interesting.

Hughes: Nobody cared much about sleep?

Barker: Well, the only time when the cyclotron was available for people
in biology was after midnight. The experiments had to be of
rather short duration, too, because the half -life is only twenty
minutes or so. And even if you got a reasonably strong radiation
sample, it didn't last more than a few hours. You had to be all
set and have rather quick methods of analysis and hope the
results would be useful. The experiments often didn't have to be
very refined in order to show novel results.

Hughes: This was the sixty-inch cyclotron in Crocker Radiation Lab?

Barker: Yes.

Hughes: Talk a little bit about what that was like.

Barker: Well, I didn't have a great many contacts directly with the

cyclotron. It was a big box for me where they put in something,
left it for a time, and it came out radioactive.

Hughes: And they handed it to you at that point?

Barker: Well, Kamen was the one who always handled the radioactive things
up until the stage that they were free of other radioactive
materials .

Ballou: You started with barium carbonate, correct?
Barker: Yes, we started with barium carbonate.

Penhoet: So how would you get the stuff to your lab? Would someone
deliver it to you?

Barker: Oh, we did the experiments up there.

Penhoet: Oh, you did the experiments right on the site?

Barker: Yes, they were all done in that old building, which was right at
the upper end of the campus. It used to be called the rat lab.
I guess that's gone now.

Unison: Oh, yes.

Hughes: It's been gone for a long time.

Ballou: It was an old wooden shack of a place.

Barker: It was a three- or four-story building.

Hughes: I've heard horror stories about the lack of protective shielding
and the way people handled the isotopes.

Barker: Yes, the people who prepared the isotope practically handled it
with their bare hands. [laughs]

Early Tracer Experiments

Penhoet: It must have been exciting when you got the first meaningful
result with radioactive carbonate.

Barker: Oh, yes.

Penhoet: Do you remember the first experiment that gave you a result?

Barker: Well, I don't remember the first one, but I remember some of the
experiments .

Penhoet: What were some of the early ones when you got some positive
results?

Barker: Well, we had experiments with methane bacteria in which we showed
that in some situations C02 disappears and methane is formed.
But there are other situations more commonly where there is C02
and you get methane, but you don't know just where it came from;
it came from an organic compound, an acetate, for example.

Ballou: There was the idea then that C02 never got reduced back to
anything; it was just the final stage in oxidation.

Barker: Oh, yes.

Ballou: If you could show that C02 got converted--

Barker: Yes, I think we were the first to show that C02 was really the
source of methane.

Hughes: And that was thanks to having Cu?
Barker: Oh, yes.

Hughes: I think it was after World War II that carbon- 14 became available
for non-military uses. Didn't you do the first tracer experiment
with Cu?

Barker: Well, I think so.

Ballou: Was this some experiment you were doing with Zev Hassid in
looking at thirty steps in photosynthesis?

Barker: Well, it's all a little vague in my mind at the moment.

Hughes: It was Kamen who was largely responsible for producing the C14,
right?

Barker: Yes.

Hughes: And I think you did some experiments with Kamen.

Barker: Oh, yes, we did lots of experiments. Nearly all of them started
late in the evening, seldom got going before ten or eleven
o'clock at night. And we seldom got home before five or six or
seven in the morning. You have only maybe three or four hours to
do the experiments .

Hughes: [scanning Barker's bibliography] I am trying to see what you
were doing with Kamen. Here we go. 1945: "Inadequacies of
present knowledge of the relation between photosynthesis and the
0I8 content of atmospheric oxygen."13

Penhoet: [scanning bibliography] Well, here: "Carbon dioxide utilization
in the synthesis of acetic acid [by Clostridium-
thermoaceticum] . "'*' That was with Kamen.

Barker: Yes, that probably was.

13 M. D. Kamen and H. A. Barker. Proceedings of the National Academy of
Sciences 1945, 31: 8.

"• H. A. Barker and M. D. Kamen. Proceedings of the National Academy of
Sciences 1945, 31, 219.

Penhoet: Another one with Kamen: "Carbon dioxide utilization in the
synthesis of acetic and butyric acids [by Butyribacterium
rettgeri] . "15 So you really were working out the [metabolic]
pathways .

Barker: Yes.

Penhoet: Hey, here's a great one: "Storage of dried fruit."16 [laughs]
Well, you did a few practical things.

Barker: Well, during the war I got quite extensively involved with people
in the food technology department, doing this, that, and the
other thing.

Hughes: You did some work on sugar metabolism.

Penhoet: Oh, sure, you did a lot of work with sucrose, with phosphorylase,
with enzymatic synthesis of disaccharides, etcetera, with Zev.

Barker: Yes, I know.

Penhoet: Well, you spent some summers working at the C&H sugar factory,
didn't you?

Barker: Oh, I was there once or twice, yes. I can't even remember what I
did there. [laughter]

Barker: The Kamen research 1 remember best was getting up there and

starting about ten at night and not finishing until two in the
morning. I think that was the standard. [laughs]

Hughes: Yes, what did your family think about that?
Barker: They survived. [laughs]

15 H. A. Barker, M. D. Kamen, and Victoria Haas. Proceedings of the
National Academy of Sciences 1945, 31:355.

16 E. R. Stadtman, H. A. Barker, and E. M. Mrak, and G. Mackinney.
Industrial and Engineering Chemistry 1946, 38:99.

Wendell Stanley

[Interview 2: January 7, 1999 ]17

Hughes: Dr. Barker, Wendell Stanley came to Berkeley in 1948, and my
understanding is that he came with the idea of uniting the
various groups that were doing biochemistry around the campus.
Do you remember anything about his arrival on campus and his
agenda for biochemistry?

Barker: I'm afraid I don't remember very much about the early period

except that it gradually became apparent that his interest in the
organization was somewhat different from ours.

Hughes: How did your views differ?

Barker: Well, I had been associated with the College of Agriculture for a
long time. Professor Hoagland was head of ag nutrition, and I
was in that for some time, and we had become accustomed to
dealing with people in agriculture and were quite comfortable, so
any change was obviously going to be somewhat different and
perhaps more complicated.

Hughes: What are your impressions of Stanley as a personality?

Barker: My recollections are somewhat vague about him, but I think he
probably wanted to have authority over it all. Perhaps he had
been told before he came that the [biochemistry] building [now
Stanley Hall] would be part of his domain. So he seemed to be
somewhat unhappy when people didn't consult him if any changes
had to be made and so on.

Hughes: He ran into quite a bit of resistance.

Barker: Yes, he ran into resistance from time to time.

Hughes: You were in the same building with him for a while?

Barker: Yes.

Hughes: How did that work out?

Barker: Well, we didn't see each other very much, except at the Christmas
parties and that sort of thing.

17 The second interview session has been rearranged for better
topicality.

Hughes : So you pretty much carried on as you always had carried on?

Barker: Well, Stanley was on the top floor and he had a fairly formal

arrangement. He had a secretary who you went to see before you
could see him, and so we didn't talk to him very much. Sometimes
he would initiate something, but by and large, we disregarded him
most of the time. [laughter] He was probably not too pleased
with that. We were in different colleges, too. He was in
Letters and Sciences and we were in Agriculture. Somehow some
arrangement was worked out so that we didn't have too many
contacts. I'm sure that he was in somewhat of a difficult
position, too. He was not interested in what we were doing; we
were not interested in what he was doing. [laughs]

Hughes: The biochemical tie wasn't strong enough.
Barker: No.

Hughes: [UC President] Robert Sproul was convinced that Stanley should
reunite biochemistry—you and the other groups around campus.

Barker: Which he didn't do; it fell apart.

Hughes: Yes, very dramatically it fell apart. But the disruption didn't
affect your research?

Barker: Well, not very much. I suppose there were times when it seemed a
little more difficult. I think on the whole we succeeded in
getting along all right despite having Stanley above us, in a
sense.

Hughes: He was literally on a higher level.

Barker: He was always on the top floor. We were on the third floor,
[laughter]

Interaction with Campus Groups Doing Biochemistry

Hughes: Did you have any close interactions with the other groups on
campus that were doing biochemistry?

Barker: Yes, I had some considerable interactions with some of the

younger people in the biochemistry department, which was part of
the medical school and under C. L. A. Schmidt, I think, at that
time. That was in the Life Sciences Building, too. That was on
the ground floor and we were on the third floor.

Hughes: Did you actually do some collaborative research? I have your
bibliography. Would that help?

Barker: Probably. [laughs] I think it got started originally over the
use of radioisotopes.

Hughes: Well, I know you worked with Kamen, for example.

Barker: Well, and with Sam Ruben who was in the chemistry department.

Kamen, who was in the Radiation Lab, was not directly connected
to any of the departments at the time.

Hughes: You mentioned that you also collaborated with the biochemists in
LSB.

Barker: Well, let me see if there's anything here [in my bibliography].

Well, I had collaborations, of course, with Doudoroff, who was in
the bacteriology department, and Hassid was in the same
department I was in. There were several papers that were
collaborations with Dr. Doudoroff.

Hughes: On a given topic?

Barker: Mostly on a given topic, namely sugar metabolism—various ways in
which bacteria of different sorts handled sucrose and related
sugars .

Hughes: Do you remember what you did and what he did?

Barker: Well, I must say I'm rather vague about that at the moment.

Hughes: He was a microbiologist?

Barker: Yes, he was a microbiologist. I'd known Doudoroff slightly at

Stanford, not very well; I really didn't get acquainted with him
until he came to Berkeley.

Hughes: Was he an undergraduate at Stanford?

Barker: He was probably a graduate student. Well, he was probably an
undergraduate when I first knew him, yes.18

Hughes: Did he have a biochemical approach similar to yours?
Barker: Yes, he had also studied with van Niel at Pacific Grove.

18 Doudoroff was undergraduate and graduate student at Stanford.

The Delft Laboratory of Microbiology

Orientation towards Microbial Biochemistry and Natural
History

Hughes: Van Niel's interest was in biochemical aspects of microorganisms?

Barker: Yes, right. Well, the Delft laboratory from which he came--where
I spent a year as a postdoctoral fellow—had an interest both in
the organisms themselves and in what they did in a chemical way.

Hughes: Was that an orientation that was characteristic of the Delft
School?

Barker: I think so, yes. Much of bacteriology at that time, and perhaps
still, was oriented toward medicine, and the Delft School had no
orientation at all toward medicine; it was entirely toward what
occurs in nature. The laboratory had developed from the work of
Beijerinck. He was one of the early general soil and nature
microbiologists .

Hughes: I brought you a book. Are you familiar with it?

Barker: Ah, yes. I have a copy of it somewhere.

Hughes: Dr. Barker is looking at a biography of Martinus Beijerinck.19

Barker: He was the great originator of the field of general microbiology
in the world at that time. There were some Russians, whose names
I don't remember-- [Sergei] Winogradsky and some others—who were
also instrumental in developing this field, but Beijerinck was
very important.

Hughes: Yes, and I associate with Beijerinck 's name the technology of
enrichment culture.

Barker: Yes.

Hughes: Was he the originator of enrichment culture?

Barker: Well, he was certainly one of the early people who developed it
more extensively than it had been previously.

19 G. van Iterson, Jr., L. E. den Dooren de Jong, and A. J. Kluyver,
Martinus Willem Beijerinck: His Life and His Work, Madison, WI: Science Tech,
Inc., 1983.

Hughes:

Barker:

Hughes :
Barker:

Was it more difficult to get financial support for looking at
microorganisms as aspects of nature than if you had been

interested in them as pathogens?

Well, I don't think so. It actually might be true in general,
but during that period I think it was relatively easy to get
support for fellowships and so on. Van Niel had made quite a big
impression by his discovery, particularly, of the photosynthetic
bacteria. And the Delft School was well known, and there were
various laboratories in the United States—Wisconsin and others--
where general microbiology was being pursued.

So general microbiology was a fundable area of research.
Oh, yes.

Physical Layout and Personnel

Barker: Yes, there's the building where I worked. [points to photograph
of building in Delft in Beijerinck biography] It was a
combination of house and laboratory. The higher part was the
living quarters and so on for the professor, and the laboratory
was tacked on to one lower part which doesn't really show here.
But it was on the lower building which extended along the canal
some little distance.

Hughes: What a wonderful setting.

Barker: Yes, it was a nice place. Boats were always going up and down
the canal. It was a major commercial highway in Holland.

Hughes: Did you communicate in English?

Barker: Oh, yes. All the professors there spoke very good English. A
number of the graduate students didn't, but most of them knew
some English, and I gradually learned a few words of Dutch.

Hughes: What was Kluyver like as a personality?

Barker: Well, he was a very large person and outgoing man. He was a very
good speaker. He gave lectures and made a good strong impression
on other people, I think.

Hughes: Was he available to you in that year that you were in Delft?

Barker:

Hughes;
Barker:

Hughes :
Barker:

Hughes :

Barker:

Hughes:
Barker:

Hughes :
Barker;

Yes, he was quite available. The laboratory and his house were
connected, you see. Once or twice a week he would come down to
the laboratory and talk with various students and so on, but if
anything important arose, you could always contact him rather
readily. He had a secretary that helped him, too.

He at that stage was mainly an administrator?
any bench work?

He wasn't doing

Oh, he didn't do any bench work,
long time.

He probably hadn't for quite a

How old a man was he at that point?

Well, let's see, that was in the thirties. Well, I would say he
was in his late fifties, maybe, or early sixties. He was very
approachable, and he spoke German; he spoke Dutch; he spoke
English. I remember there was an international meeting and he
greeted the people who came to the lab in three or four
languages. [laughs]

Was the lab quite a crossroads for scientists interested in
microorganisms?

Yes, quite a few people visited during the year I was there. I
can't remember now who they all were. Oh, some people from the
University of Wisconsin-- [Chester H.] Werkman, I remember
particularly. He was traveling in Europe and he spent two days
there in Delft.

But didn't do any research?

No. I'm a little vague now about whether there was anybody else
from the United States there while I was . The Dutch speak
English and German quite well. The technical people- -the
assistants who are paid to work there--didn' t speak English very
well, but I could get by all right with them with my poor German.

How big a group was it?

The laboratory itself had about six or eight, maybe ten, rooms.
Well, I can't remember in detail how many. Professor Kluyver
always had one major assistant in the labs, which van Niel had
been at one time. And when I was there, it was a man by the name
of Kingna-Boltjes , who later became professor of microbiology in
northern Holland after he left there.

Hughes: What was his area of expertise?

Barker: Well, let's see, he had done his Ph.D. on nitrifying bacteria, I
believe, yes. But generally he covered the same area that
Professor Kluyver did, although he was appreciably less
articulate and so on. Kluyver had a very outgoing personality
and spoke very well publicly as well as privately.

Hughes: Were you having scientific interchanges with Kluyver?
Barker: Oh, well, that was the main thing, yes.
Hughes: So you were talking about your research?

Barker: Yes, I was talking about my research, and other people's research
which might be related.

Dr. Barker's Research

Hughes: Did you go to Delft with a specific research project in mind?

Barker: Not really, no. I decided, as I recall, to fit in with whatever
[Kluyver] would suggest. And I remember, initially, that I was
also interested in methanogens . I got started on the methane-
producing bacteria. I'd started this in Pacific Grove, and so
that's one of the things I continued on, but initially he started
me out on some smaller project — some easier project.

Hughes: Do you remember what that was?
Barker: Well, I'd have to look up and see.
Hughes: Do you think you published on it?

Barker: Oh, yes, I'm sure it was published. Well, let me see. [skims
his bibliography]

Hughes: You were in Delft from 1935 to 1936, so presumably the
publication can be no earlier than 1936.

Barker: Yes, I think one publication was made while I was there. Let's
see here. Yes, I think the first publication that I did while I
was there was, "On the fermentation of some dibasic C<,-acids by
Aerobacter aerogenes. "20

20 H. A. Barker. Proceedings of the Koninklijke Akademie van
Wetenschappen te Amsterdam 1936, 39:674.

Hughes: Why do you suppose Kluyver gave you that project?

Barker: Oh, I don't know; it was relatively easy, I think, to get started
on.

But the main work that I did, so far as my future was
concerned, was starting on the isolation of methane-producing
bacteria. And this particular organism produced caproic and
butyric acids in large amounts. And one paper was published in
the Archives of Microbiology in '37.21

Hughes: Was that a significant contribution?

Barker: Yes, it was, because I think no organism producing caproic acid
had been known before that time, so this was somewhat novel.

Hughes: Why would that be interesting?

Barker: Well, I suppose it's interesting because caproic acid is a six-
carbon compound and butyric acid has four carbons. And I suppose
it extended the range of chemistry. Also caproic acid apparently
had some uses which butyric acid didn't have. Butyric acid and
butyl alcohol had been produced commercially previous to that
time, and the organism that I isolated was used in Delft later
on, I think, for caproic acid production—maybe something else,
but I'm not sure what now.

Hughes: What is caproic acid used for?

Barker: Well, as a compound it just has two more carbon atoms [than
butyric acid] .

Hughes: Yes, but does it have some industrial use?

Barker: Well, evidently it did have some, or Professor Kluyver thought it
might .

Hughes: You, I'm gathering, were not particularly interested in the
practical applications of this work.

Barker: No.

Hughes: You were interested in how these organisms functioned in nature?

21 H. A. Barker. "The production of caproic and butyric acids by the
methane fermentation of ethyl alcohol." Archiv fiir Mikrobiology 1937, 8:415,

Barker: Yes, but you know I was a young person getting started. To have
something that is of some interest outside of the laboratory is
also nice. [laughs]

More on Faculty Participants

Barker: Greenberg, I think, did go to San Francisco for a time.

[referring to 1946 memo re faculty participants in the graduate
group] [Paul] Kirk: his primary interest was in microchemistry .
He had interests outside the university in consulting of various

sorts. I don't remember now which group he stayed with.
[Sherburne F.] Cook I think was in Physiology. Hassid and I were
in Plant Nutrition under Professor Hoagland, and Joslyn was in
Food Technology. He was in one of the buildings along the north
side of the campus, before the big buildings right along the
street were built. Food Technology, Plant Pathology, and several
other small departments were located in that building, if it's
still there. [Harold] Tarver was in Biochemistry and Mackinney
was in Food Technology, Doudoroff was in Bacteriology, and
[Samuel] Lepkovsky was sort of by himself up the canyon.29

Hughes: At LBL [Lawrence Berkeley Laboratory]?

Barker: No, there was an old laboratory--! 'm not sure if it still exists
--which was above the Biochemistry Building up the canyon a ways.
There was a road that went up there past the tennis courts and
the swimming pool and ultimately the road turned up and went up
to the Radiation Lab up on the hill, and there was a whole
building there that was very antique.

Hughes: And no longer exists?

Barker: Well, he got along there until he retired and after he retired I
think it was probably torn down.30

Student Participation

Hughes: I read somewhere in those documents that there were eleven

different departments represented in the Program in Comparative
Biochemistry.

Barker: Oh, yes. It presumably took in most of the biology departments,
and some of them I think probably never had any students in this
area and some of them had several. I think Food Technology had
several students, Bacteriology had some, Plant Nutrition had
some. Well, there may have been some others, too. It was not a
large group.

Hughes: Was there ever a problem in the different agendas that I presume
both students and professors brought to the group? A field such

29 Dr. Ballou notes: "Lepkovsky was in Nutrition, worked on poultry
husbandry and had a lab on what is now called Cenetennial Drive. The building
is still there and is devoted to Atmospheric Aerosol Research."

30 Louise Taylor believes the building may have been used thereafter by
Poultry Husbandry and then torn down recently.

as Food Technology, for example, has a practical orientation, but
what you were doing was very basic research. It worked to have
an umbrella group?

Barker: Well, when it came to a Ph.D. examination, one put people on the
committee who were appropriately there. [laughs]

Hughes: So it was a large enough group that you picked and chose
according to the needs of the dissertation committee?

Barker: Well, the students really chose us rather than we choosing the
students. It was designed for students who didn't quite fit in
the more specialized requirements of a particular department.
They were required to take a variety of general courses-
bacteriology and biochemistry- -and then they always took some
more specialized courses — in physiology, and so on.

Hughes: Did all of your students work through this graduate group?

Barker: No, I had some students that got degrees in microbiology, which
was also an interdepartmental group. Well, [our comparative
biochemistry] group was really designed to take care of students
who for one reason or another didn't fit in with a particular
department, whose interests in terms of the standard departments
were sort of interdepartmental. It was not very large. I think
we never had more than six or eight students at one time, but it
lasted for a number of years.

Hughes: You will see in those documents a listing of the course
requirements.

Barker: Here we go. So we had Chaikoff , Cook- -what department was he in?

Hughes: Is that Sherburne Cook?

Barker: It's S. F. Cook.

Hughes: I'm almost sure he was in the Department of Physiology.31

Barker: I think he was, too. He wasn't a very prominent member [of the
Program in Comparative Biochemistry]; I don't think he was there
very long, either.

Hughes: I know his name because he, like you, was one of the early users
of radioisotopes . But not as early as you were.

31 Cook joined the department in 1928 as an assistant professor.

Seminars

Hughes: I also noticed some reference to seminars that were supported by
the graduate group as a whole. Do you remember that? These
seminars were different from those that presumably each
department sponsored.

Barker: Well, we did for a number of years have seminars in general

microbiology. I think Mike Doudoroff and Roger Stanier were in
charge of that at one time. I think we would each take turns
finding people who would be willing to talk.

Hughes: Those seminars were for the Department of Bacteriology or were
they for the Program in Comparative Biochemistry?

Barker: Well, I think it was the Program in Comparative Biochemistry.

The senior people in bacteriology were rather medically oriented,
except for the people who had come from van Niel's laboratory,
and so they, instead of combining with the medical people, had
their own seminar series which was on nonmedical subjects.

Hughes: So that was another purpose of the program; it allowed you to
explore subjects that might have been a bit more difficult to
explore, at least on a seminar basis, in specific departments.

Barker: Oh yes, I think so.

Hughes: Was there anything else that the Program in Comparative
Biochemistry allowed one to do?

Barker: Well, it mainly functioned to handle graduate students who did
not want to get directly involved in the medical biochemistry
departments, and there were quite a lot later on. I don't know
if any of them are here. Let's see, there are one, two, three,
four-- [continues counting to fifteen before interrupted]

Hughes: Are you counting up students who--

Barker: Who I had.

Hughes: In the Program in Comparative Biochemistry?

Barker: Yes.

Hughes: You wrote in an article in the Annual Review of Biochemistry that
seventy-five students majored in comparative biochemistry.32

Barker: No, those were not all mine because this included people in the
bacteriology department under Stanier, Doudoroff, and [Edward]
Adelberg, and I think there were maybe even one or two in other
departments.

Hughes: That's quite a number of students.

Barker: Well, it was the time when general microbiology, and biochemistry
related to it, flourished in Berkeley.

Hughes: Those years were the high points?

Barker: Yes. Doudoroff was very much interested, for example, in

problems related to sucrose and so on, and I was involved in this
somewhat also. We got a lot of publicity on bacterial synthesis
of sucrose, not that it ever had any practical application; it
was theoretical.

Hughes: Yes, I saw some newspaper articles about the sucrose work.33
Barker: Yes, there was some publicity that got out in the press.
**

Hughes: Do you remember why the name Program in Comparative Biochemistry
was chosen? Why wasn't it just the Program in Biochemistry?

Barker: Well, there was a biochemistry department, you see, and we had to
distinguish ourselves. It was in the medical school at the time.
That was one of the slight complications. Also, Greenberg, the
chairman of the medical school department, I think was a little
sensitive about having another group with a name that was too
similar. [laughs]

Hughes: What you were doing in your research was certainly comparative
biochemistry, is that not true?

Barker: Well, we used various organisms, yes, mostly bacteria.

32 H. A. Barker, "Explorations of bacterial metabolism," Annual Review of
Biochemistry 1975, 47:1-33.

33 Barker's papers in the Bancroft Library are a rich source for all
aspects of his career.

Hughes: But that wasn't really the agenda of the group as a whole?

Barker: Well, it wasn't actually a very big group. There were only about
half a dozen people [faculty members], I think, who ever made use
of this. It was mostly the students. I think my students, I
think Hassid's students, and Chaikoff's students made use of
this. And then some of the people in Food Technology-- Joslyn, I
think .

Teaching

Course in Soil Microbiology

Hughes: Do you remember what courses you taught?

Barker: I taught primarily two courses during this period. I was brought
to the university to teach soil microbiology. Initially, when I
first came, I think there were only two students that took the
course. Dean Lipman had taught it several years in his spare
moments, but he didn't do a very systematic job of it. He mostly
came around and got people started on some simple experiments and
perhaps once or twice during the semester gave a lecture or
something. [laughs] It was a very informal course, and there
were very few students. I think one reason I was hired was to
take care of the students in Plant Nutrition that needed to know
something about soil microorganisms. So after I came it was a
required course in the soils curriculum.

Hughes: So you had a lot of students?

Barker: Well, not a lot because it wasn't a big group, but on the order
of fifteen or twenty, or maybe it was as high as thirty
sometimes.

The van Niel Approach to Biochemistry

Hughes: Were you more or less following van Niel's approach?

Barker: Well, to some extent, because that's what I knew. You see, I had
studied with van Niel and I had been a year in Delft with
Professor Kluyver in the laboratory where van Niel had
originated. But this course in biochemistry was not primarily
soil microbiology; it was only partially so. We used various
sorts of material—plant material and perhaps some bacteria--to
set up experiments that could be done rather easily.

Hughes: What was the second course you taught?

Barker: Well, some of the people in bacteriology also had been students
of the same people in Stanford that I [had studied with] ,
particularly van Niel at Pacific Grove, and so we had him in
common.

Hughes: Was that Doudoroff?

Barker: Yes, Doudoroff, Stanier, and Ed Adelberg.

Hughes: Oh, Adelberg had been with van Niel, too?

Barker: Well, yes, I think he'd been there one summer.

Hughes: So they were all imbued with the van Niel approach to
biochemistry?

Barker: I think so.

Hughes: What would you call it? Microbial biochemistry?

Barker: Yes. Ed Adelberg was a little more connected to genetics, but

the others were primarily interested in biology and biochemistry.

Hughes: Stanier and Doudoroff were in Microbiology, is that correct?

Barker: They were in Bacteriology, primarily. K. F. Meyer was head of
that department for many years.34 And later on perhaps Stanier
may have been head of the department for a short time—two years.

Hughes: Where was Adelberg?

Barker: He was in Bacteriology, too. He later on left and went somewhere
in the East—to Yale, I think.

3* See the oral history with Karl F. Meyer in The Bancroft Library oral
history series on public health at Berkeley.

Barker's Teaching Style

Hughes: Did you like teaching?

Barker: Oh, I don't think I was ever as enthusiastic about teaching as,

for example, van Niel was, who was the person who really inspired
me in the area of bacteriology.

Hughes: Could you describe your style of teaching?

Barker: Oh, 1 don't know. In the bacteriology course for agriculture

students, I suppose to some degree I tried to imitate van Niel--
not very well, however. But at any rate, we had a laboratory,
and we had, I think, two lectures a week that more or less
covered the general explanation of what we were trying to do.

Hughes: And you stood up and gave a formal lecture?

Barker: Yes. Well, it wasn't very formal. It was done in a different
room; we had a regular lecture room. From time to time I would
talk about the internal chemistry of microorganisms and so on. I
would do that in the laboratory. The formal lecture part that
was done in a lecture room generally covered the classical
aspects of soil microbiology.

Hughes: What would you classify under classical? [laughter]

Barker: Well, I had studied with van Niel in Pacific Grove, and we

learned a lot about the biochemical systems bacteria utilized.
Then I was in Holland for a time, and I enjoyed lectures from
Professor Kluyver that were in Dutch. For a time 'I had a little
difficulty following the Dutch. It was all very nonmedical,
talking about soil transport issues.

Hughes: And emphasizing the biochemical aspects of the microbial world?

Barker: Yes, well, why they're important, you see, is because they
changed the chemistry.

Hughes: In medical school, I suspect that you'd get quite a different
emphasis.

Barker: Oh, yes, they'd be talking about disease. I was not interested

in that at all. I wouldn't cover it unless it was something that
just happened to be related.

Hughes: Did it make a difference to you whether your students came from a
microbiological outlook and background as opposed to a
biochemical?

Barker: Well, the students that I had were almost entirely students in
the soils curriculum and they had a fairly modest amount of
chemistry. They must have had some biochemistry, [but] they
weren't very high-powered. The more complex and scientific
aspects of the chemical reactions in bacteria I got into with
Ph.D. students.

Hughes: They presumably came to you because they also were interested in
the biochemical aspects.

Barker: Yes, they came from various other departments. I guess most of
my students actually got their degrees in agriculture.

Research on Anaerobic Bacteria

Hughes: Do you have a favorite piece of research?

Barker: Well, at one stage I'd been interested in the transformations of
some of the anaerobic bacteria that I worked with, particularly
Clostridium kluyveri, which I named after the professor I worked
with in Holland.

Hughes: Oh, you named it!

Barker: Yes, I was the one to isolate it. It was isolated from the mud

of a Dutch canal. [laughs] I published quite a number of papers
dealing with various aspects of its biochemistry which was rather
interesting. I worked on that off and on for a number of years
and a couple of my students got Ph.D.s working in this area.

Hughes: Is there anything more you want to say?
Barker: I don't think so.
Hughes: Thank you.

Transcribed and Final Typed by Amelia Archer

TAPE GUIDE--Horace A. Barker

Interview 1: December 21, 1998

Tape 1, Side A 1

Insert from Tape A, Side A 12

Tape 1, Side B 15

Insert from Tape 4, Side A 20

Interview 2: January 7, 1999

Tape 2, Side A 26

Tape 2, Side B 36

Tape 3, Side A 38
Tape 3, Side B not recorded

Interview 3: January 14, 1999

Tape 4, Side A 50

excerpts moved to page 12 and 20 55

Tape 4, Side B 59

APPENDIX

H. A. Barker correspondence, B-12 research 62

U.S. Patent 3,037,016 66

"Explorations of Bacterial Metabolism," H. A. Barker, Ann. Rev.

Biochem. 1978. 47:1-33 75

Horace A. Barker, Curriculum Vitae 108

Obituary, UC Berkeley Media Release, January 8, 2001 110

Memorial Service Program 113

R: SQUIBB &_ SONS 745 Fifth Avenue, New York 22, N. Y. PLaza 3-2900

Division ol OLIN MATHIESON CHEMICAL CORPORATION

THE PRICELESS INGREDIENT IS THE HONOR AND INTEGRITY OF THE MAKER

ROLAND J. DAIIL

VICE-PRESIDENT

DIRECTOR or RESEARCH AND DEVELOPMENT

May !>, 1959

Professor K. A. Barker

Department of Agricultural Biochemistry

337 Biochemistry and Virus Building

University of California

Berkeley, California

Dear Professor Barker:

As a result of the interest of members of our
technical staff, my attention has been called
to your experimental program studying the
coenzyme forms of the B]_2 group of vitamins.
We are very interested and impressed with your
findings on what may prove to be the natural
form of these important nutritional factors.
We are, of course, primarily interested in the
therapejatic value of the B^^S^enSyjTies and would
llke^tcT "inform yovfof "our" intention to apply
for licenses under any patents you and your
university will obtain.

May I assure you of the willingness of our
laboratory group to supply you without obliga
tion, in the near future as in the past, with
any materials that will be useful to you in
carrying out your program.

truly yours

•t- '

May 6, 1959

Mr. R. M. Underhill
2l*0 Sproul Hall
Campus

Dear Mr. Underhill:

I am enclosing a letter from R. J. Dahl,
Vice-president of Squibb and Sons, which indicates
that Squibb is interested In obtaining a license
for B£^ t&Snzyme production under the patent for
vhich I recently applied. Enclosed is a copy of
my reply. I assume that you will handle any
further business arrangements.

Squibb has been very helpful in providing
us with materials useful in our research on the B12
. coenzymes. No other group has been so helpful with
the scientific aspects of our work nor so easy to
deal with, ,00 I would look with favor upon a license
;?. application by this Company. /

Recently I had a telephone conversation
with Dr. -Otto Benrens of. the Lilly Research Labora-
t^tories, and meatloa^'"*l»t I. had .applied; for X^
.- -^patent coyering the .fii^'coenzynes'. " He^ejcpres'sed
-'i -Interest and said he^ expected .tfo'contaxtVyou before

With best regards,

.:.'•... i';*

Sincerely yours,

^:*!' ••'. . i. ?V^!Wr2-

': >^'HAB:a; ';^^,"---- . H. A. Barker

October 20, 1959

Mr. Robert M. Underbill

Secretary and Treasurer of the Regents

University of California

615 University Hall

Campus

Dear Mr. Underbill: Re: Vitamin BI? Compounds

I was glad to receive copies of your letters to the
various pharmaceutical companies indicating the present status
of the negotiations concerning licensing under my patent
application.

At present there is only a single publication dealing
vith coenzymes possessing vitamin Bi2 activity. This is a
paper published in the Proceedings of the National Academy of
Sciences (U.S.), k£, 521-525 (1959), a copy of vhich is enclosed.
This paper describes some properties of the partially purified
coenzymes, but does not describe the methods of purification
nor the final isolation of the crystalline compounds. The
first paper giving this information will be published in the
Journal of Biological Chemistry, probably in January 1960.
I expect that any further foreign patent applications should
be made before this date.

I should mention that there is now considerable
evidence that our "vitamin. B^xajcoenzyme" is approximately as
gffectiye_as vitamin BIS itself in the treatment of pernicious
anemia and in the nutrition of chickens. We are still looking
for situations in which the compound may be more effective than
vitamin Bi2 as a nutrient or chemo therapeutic agent for animals
or man. There is extensive Interest in the coenzyne as a
reagent for biochemical and medical research and there trill
certainly be some commercial market for these purposes at least.

Yours sincerely,

HAB:a H. A. Barker

United States Patent OiSce

Patented May 23, 1C 52

3,037,015
Bu COENZYMES AMD PROCESSES FOR

PREPARING THE SAKE

Horace A5bcrt Barker, Berkeley, Calif., assignor to Tbc
Regents of Til Unlvcrsify cf California, Ecrkelcy,
Calif.

No Drawing. Filid Apr. 13, 1959. Scr. No. 805,728
16 Claims. (C!. 260—211.5)

This invention is concerned generally with novel co-
enzymes having vitamin B12-activity and v/ith procedures
for preparing them. More particularly, the invention
relates to the new chemical compound, coenzymc BU.
and novel vitamin 3I3-activc analogs thereof, and to novel
processes for producing these new coenzymc BI3 com
pounds by controlled bacteriological synthesis and degra
dation of the resulting bacterial cell material. These
novel vitamin Bu-active cocnzyme Bla compounds, which
can be characterized by their property of activating the
enzymatic conversion of glutamatc to mesaconate via
B-mcthylaspartate, are valuable as feed supplements and
for the treatment of nutritional diseases. They are fur
ther valuable as growth-promoting agents and in biologi
cal investigations of essential enzymatic reactions involved
in metabolism and maintenance of health.

Vitamin S\-, which possesses marked and effective
action in tic therapeutic treatment of Addisonian per
nicious anemia and other ciacrocytic anemias, may be
chemically represented as follows:

HjN-OC-CHrCHi

H.N-OC-CH,
Mo

Mo
JS-CHrCO-NHi

CHrCHrCO-NHi

(bHrCHrCO-NHi

Consistent with this structure, vitamin B:3 and vitamin
Bu-like compounds (which differ from vitamin Bu in
that the cyano radical attached to the cobalt atom is
replaced by a different grouping, and which are convert
ible to vitamin B13 per se by treatment with cyanide ion)
are called cobalamins; vitamin B13 itself is referred to
as cyanocobalamin; vitamin B:3-active compounds, in
which the 5,6-dimethyIbenzimidazole moiety present in
the cobalamins (which acts as a bridge between the ribose
and corphyrin portions of the molecule) is replaced by
another nucleotide base, are herein referred to as vitamin
BI3 analogs.

The vitamin B13-active coenzyme Bu compounds, sub
ject of the present invention, are structurally similar to
vitamin BU and those of its cyano analogs, in which the
nucleotide base is a benzimidazole compound (such as
benzimidazole, 5,6-dimcthylbenzimidazole and 5-bydroxy-

bcnzimidazole) but differ from these vitamins B1; com
pounds in lacking a cyano group and in possessing an
adcTiine moiety attached to the corpliyria portion cf the
molecule. These novel vitamin Bi3-active cocrr.yme 3I;

S compounds as, for example, coenzymc Bi3 (which con
tains 5,6-dimcthylbcnzimidazole as the nuclcotic: ba:c
and which is convertible to vitamin BI3 by treatment with
cyanide ion), benzimidazolc-cocnzyme BI:. bydroxybcn-
zimidozole-cocnzyme B13, and the like, are potent gro-.vth-

10 promoting agents, and are valuble in nutrition and in
the treatment of nutritional diseases. As the first co-
enzymes of the vitamin Bu-activc group, the?e aev/ co-
enzyme BU compounds are particularly valuable to bio
chemical and medical research workers in connection

15 with investigations of essential enzyme reactions involved
in normal metabolism and maintenance of health, as well
as in studies of abnormal metabolic processes charac
teristic of certain diseases. The vitamin B13-active co
enzyme BU compounds can be characterized by their

20 property of supporting the growth of Ochromonas mal
hamensis and by their ability to activate the enzymatic
conversion of glutamate to mesaconate via /9-methyi-
aspartate.
These coenzyme Bla compounds are produced by fer-

25 menting, with a vitamin BI3-activity producing micro
organism, an aqueous nutrient medium containing, where
indicated, the benzimidazole precursor corresponding to
the coenzyme B13 compound desired. As the vitamin B:J-
activity producing organism, selected strains of micro-

30 organisms belonging to the Schizomycetes arc ordinarily
employed, particularly certain strains of the gcaus Strep-
tomyccs, the gcaus Bacillus, the genus Propionibac'.crrjm,
the genus Alcaligenes, the genus Pseudomonis, the genus
Mycobacterium, and the genus Clostridium, prefcribly

35 strains selected from the species Strcptomyces griseus,
Streptomyces albidoflavtu, Streplomyces frcdine, Strepto-
myces venezuelae. Bacillus mesaterium, Propionibac:-:-
rium shermc.iii, Propionibacterium freudenrcichii, Pro
pionibacterium arabinosum, Alcaligenes faecalis, Pscu-

*" domonas aeruginosa, Pseudomonas fluorescent, Pssu-
domorMS iumichroma, Mycobacterium smegmatis, Clo:-
trulium tctanomorphum, and the like. The benzimid
azole precursor corresponding to the coeazymc 3,- com
pound desired is ordinarily incorporated in the autrier.l

45 medium prior to fermentation, although many vitamin
Bu-activity producing genera (for example Strcptomyces,
Bacillus, Propionibacterium, Pseudomonas and Mycobac
terium) produce substantial yields of coenzyms B13 per se,
utilizing nutrient mediums not containing added 5,6-di-

50 methylbenzimidazole.

The vitamin Bj3-activity producing microorganisms uti
lized in producing the new coenzyme B13 compounds ars
conveniently selected by testing their fermentation broths,
using the protozoan Ochromonas malhamensis as the

65 assay organism. A culture of the microorganism under
investigation is diluted, plated out on a solid nutrient
medium, and incubated to produce a considerable number
of single-ceil colonies. Individual colonies picked for
inoculum development are separately grown in liquid

60 nutrient mediums supplemented with cobalt nitrate at a
concentration of 1 p.p.m. and with 5,6-dimethyroeazimid-
azole at a concentration of 0.0001 molar in suitable ves
sels and incubated cither in presence or absence of or.y-
gen, depending on the requirement of the organism. The

65 fermentation broths are heated (where necessary) to co
agulate the cells, and the resulting solution is assayed for
Ochromonas malhamensis activity.

The basal medium employed for the growth of Ochro
monas malhamensis, which eliminates non-specific growth

70 stimulants present in certain crude extracts to which the

3,037,016

organism has proved susceptible, has the following com
position:

Casein hydrolysate g__ 5

Glucose g 10

Diammonium hydrogen citrate g 0.8

KH,POi g— 0.3

MgS<V7H3O g._ 0.2

CaCOj g__ 0.15

Ethylencdiamine tctra-acetic acid mg 50

MnSOfHjO mg._ 61.5

ZnSo4-7H:O mg-_ 110

FeSO<-7H3O mg-- 10

CoSO^HsO mg._ 3

CuSo4-5HaO mg__ 0.4

H3BCS mg— 0.6

KI mg.. 0.01

N.ijMoO4*2HjO ___________ ... .mg 50

DL-tryptophane mg 100

DL-methionine mg__ 200

L-cystine mg 100

Choline chloride mg-_ 2

Inositpl mg._ 10

p-Aminobcnzoic acid mg — 1

Thiamins mg— 2

Biotin jig__ 10

Twecn 80 l mg— 1

pH adjusted to 5.5.
Distilled water to 200 ml.

1 A polvoxyetbylene derivative of sorbltol mono-oleate gait-
able for use In mlcroblologlc.il cultures.

The test organism is maintained in the basal medium
diluted 1 part of medium to 5 parts distilled water and
supplemented with 0.2 m^g. cyanocobalamin/ml. The
diluted medium is dispensed in 10 ml. amounts into 50
ml. conical flasks, which are then plugged and sterilized
by autoclaving for 15 minutes at 10 Ib./in.3 pressure. The
organism is transferred in this medium at 5-day intervals,
and incubated in a cabinet at approximately 27* C., 1
ft. below a 60 w. tungsten filament lamp. After 5 days'
incubation under these conditions the cell population
density in the cultures reaches approximately 5,000,000
cells/ml. For inoculum, a 5-day culture is diluted 1:10
with sterile basal medium diluted 1:5, and 0.5 ml. is
added to each assay tube.

a given microorganism is potentially capable of synthe
sizing vitamin Bi3-active coenzyme Bu compounds, as
well as the amount of vitamin Bi3-acn've substances cor.-
tr-ined in the cells and fermentation broth obtained when
said microorganism is used to ferment an aqueous nutri
ent medium.

The bacteriological production of the presently-in
vented coenzyme BI3 compounds is conducted utilizing
aqueous nutrient mediums ordinarily employed in the
propagation of microorganisms. The usual nutriecls
include an energy source, a carbon source, a nitrogen
source, inorganic salts, and growth factors when required.
It is preferred to supplement the medium with a source
of cobalt, ouch as cobalt nitrate; in addition, the nppro-

jj priate bcnzimidazole precursor (e.g., benzirnidazolc; 5-
hydroxybcnzimidazole; 5,6-dimethylbenzimidazole) is or
dinarily incorporated in the medium, although no added
precursor is required for producing cocnzyme B!3 per EC
using many vitamin B:3-activity producing genera-, cs

2Q noted hereinabove. The carbon and energy can be pro
vided by a carbohydrate such as dextrose, maltooc, xylose,
invert sugar, corn syrup, and the like, and by amino
acids such as glutamic acid (in the form of its neutral
salts). The nitrogen can be provided by an ammonium

25 salt, amino acids, proteins or protein degradation products,
obtained from proteins such as soy beans, oats, yeast,
yeast extracts, casein, meat, blood meal, protein meat and
bone scrap, salmon meal, fish meals, fish solubles, dis
tillers solubles, and the like. If desired, the microorgan-

20 isms can be propagated using proteins or protein degrada
tion products without any carbohydrate being present in
the medium, in which case the proteins serve as the source
of energy, carbon and nitrogen required by the micro
organism.

The aqueous nutrient medium is sterilized and inocu
lated with a culture of the selected microorganism strain,
and the mature is incubated underjfonditions appropriate
to the particularjaKroorgan ism employed. Since the co-
' enzyme" Bj3 compounds are extremely sensitive to de

composition on exposure to visible light, all operations in
volved in the production of these compounds are con
ducted in the substantial absence of light. It may be
noted that coenzyme BJ3 compounds arc not obtained in
accordance with the methods utilized heretofore for ob
taining vitamin BJ3 compounds, since those methods not

Assays are set up in 19 x 150 mm. optical matched 45 only failed to provide effective protection from light,
Pyrex test tubes. A standard solution of cyanocobalamin but also conventionally involved treatment with cyanide
containing 0.2 m/jg./ml. is added to paired tubes at levels ion and/or acidification to pH 3, thus precluding the
of 0.25, 0.5, 1.0, 2.0 and 4.0 ml. Test extracts of fcr- preparation of the coenzyme which is highly unstable
mented broth are added to paired tubes at the same levels in the presence of cyanide or acid. The fermentation is
(following a preliminary experiment to determine wheth- 50 allowed to proceed for a time sufficient for the bacteria!

er the broth has any Ochromonas malhamensis activity
and the approximate value of this activity), and water is
added to the tubes to bring their fluid content to 4 ml.
To each of the tubes is thea added 1 ml. of the undi
luted basal medium, the tubes are plugged with cotton,
and autoclaved for 10 min. at 10 lb./in.J pressure. The
tubes are then cooled, inoculated with 0.5 ml. of the 5-day
diluted culture referred to hereinabove, placed in a shak
ing machine in an incubator at 29* C. and shaken in
darkness for 72 hours. The tubes are then autoclaved, 5
ml. water are added to each, and the growth in each tube
is determined turbidimetrically in a Klett-Summerson
colorimeter using a 540 millimicron filter.

Since the size of the inoculum is constant for each

cells to reach maximum growth, at which time the fer
mented mixture is centrifuged or filtered, the supernatant
solution is discarded, the cellular material is recovered as
a paste and subjected to degradation to produce the co-

55 enzyme Bu compound. Alternatively the fermented
mixture is heated or allowed to undergo lysis, thereby
producing a solution of the cocnzyme B13 compound in
Ihe fermentation broth; avoidance of cyanide and/or acid,
and protection from light are essential in this operation as

60 well as in all subsequent treatments if the coenzyme C,3
compound is to be obtained. The former method, where
the cells are separated from the fermented mixture and
then subjected to degradation, results in the production
of a relatively concentrated aqueous solution of the co-

tube, the growths obtained in the control and test cultures 05 enzyme Bn compound which is substantially free from

are proportional to the concentration of vitamin BI»-
active substances contained therein. Comparison of tbo
growth of the test culture with that in the controls gives
a quantitative measure of the concentration of vitamin

impurities present in the original broth; the latter method,
which produces a relatively dilute and impure solution of
the coenzyme in the whole broth, has the advantage of
avoiding the difficult separation of the cellular material

Bu-active substances (expressed as m^g. of cyanocobal- 70 from the broth,

amin/ml.) in the test cultures and, by a simple calcula- The degradation of the cellular material (where the

tion, the precise content of vitamin B13-active substances cell paste is separated from the broth) is conveniently

in the fermented broth taken from the original fermen- conducted by heating the diluted aqueous cell paste prcf-

tation vessels. erably at a temperature within the range of approximately

From the above test, it ia possible to determine whether 75 70-100' C., although higher or lower temperatures may

3,037,010

be employed if desired; the heating is continued for a
time sufficient to coagulate the cellular material, e.g.,
about 2 to 20 minutes at 100' C. Alternatively, the
cells are subjected to the action of an alcoholic solution
as, for example, a solution of a lower alkanol such as .
methanol, ethanol, propanol, and the like, having a con
centration in water within the range of approximately
70-100%. It is ordinarily preferred to mix the aqueous
cell paste separated from the fermented mixture with
enough ethanol to give a final ethanol concentration of .-
about 80%. Irrespective of the method utilized in coagu
lating and precipitating the cellular material, there is
obtained, following separation of precipitated cells, a
solution of the coenzyme BU compound; this solution
(where alcohol is present) is then subjected to distillation lg
in vacuo, thereby evaporating the alcohol. The aqueous
solution of the coenzyme is then passed through a cation
exchange resin (preferably a sulfonic acid type resin
such as Dowcx-50, 8x, manufactured by the Dow Chem
ical Co.) in the sodium form, thereby absorbing cationic go
substances from the solution; the eluate is then passed
through an anion exchange resin containing quaternary
ammonium groupings (such as the Dov/ex-2, 8r, resin
manufactured by the bow Chemical Co.) in the hy
droxide or acetate form, thereby absorbing anionic sub- 25
stances including acidic nuclcotidcs and amino acids.
The resulting cluates and washings are combined and
adjusted to pK 6.5-7.0, conveniently with 1 N acetic
acid solution.

The resulting solution is then extracted with an hy- JQ
droxylatcd organic solvent characterized as being partially
immiscible with water as, for example, a phenolic solvent
such as phenol or cresol, an alkanol such as butanol,
amyl alcohol, and the like, or a mixture of such by-
droxylated solvent and a hydrophobic solvent, as for 35
example a hydrocarbon solvent such as benzene, toluene,
a chlorinated hydrocarbon solvent such as ethylenedichlo-
ride, trichlorethylene, and the like. Tnere is added to
the hydroxylated organic solvent extract a lower ketone
such as acetone, methylethyl ketone, and/or a dialkyl 40
ether such as ethyl ether, dipropyl ether, and the like,
thereby forming an upper organic layer containing the
hydroxylated organic solvent and a lower aqueous phase
containing the coenzyme. It is ordinarily preferred to
utilize phenol as the hydroxylated organic extracting 45
solvent and to add to the phenolic extract a 1:3 mixture
of acetone-ether. The organic layer is extracted with
water until the aqueous extract is virtually colorless. The
combined aqueous extracts are washed with a substan
tially water-immiscible solvent such as ether to remove 60
hydroxylated organic solvent, and the aqueous layer is
distilled in vacuo, thereby evaporating ether remaining
in the aqueous phase and forming a relatively concen
trated aqueous solution of the coenzyme BJJ compound.

This solution, the color of which (depending on the 65
concentration of the coenzyme Bw compound) varies
between orange and red, is then passed into a column
of a weakly acidified (pH approximately 3) cation ex
change resin, preferably of the sulfonic acid type, in the
mixed sodium-hydrogen form; as the resin, it is preferred 60
to use a copolymer of styrene in which the divinylbenzene
component is between 1 and 4%. (A commercially-
available resin of this type is manufactured by the Dow
Chemical Co. under the trade name Dowex 50W-2*.)
Resins containing higher cross-linking have proved less 66
satisfactory. The resin is adjusted to pH approximately
2.5 to 3.5 (mixed Na+ — H+ form) at which pH coenzyme
B13 compounds have a positive charge and are adsorbed
on the resin; the free vitamin B1S compounds in the
cyano form (which are substantially neutral) are not 70
appreciably adsorbed at this pH. The solution of co
enzyme B12 compound is rinsed into the column and the
coenzyme is adsorbed to form a thin orange-red band
at the top of the column. (All observations as to color
of solutions and of bands of material adsorbed on col- 75

umns are made with dim light and with minimal time
of exposure to avoid substantial decomposition of thi
coenzyme Bu compound.)

The resin column is then subjected to differential elu-
tion with buffer solutions of gradually decreasing acidity
containing Na+ as the competing ion. It is ordinarily
preferred to employ solutions of sodium acetate v/ithin
the range 0.01 to 0.2 molar, although other sooium baJcr
salts, e.g., sodium propionate, sodium phosphate, ar.d
the like, may be used if desired. The initial elutioa is
preferably conducted with 0.03 M sodium acetate at pK
4.6 to 4.8; when about 5-6 column volumes of eluate
have been collected, the eluting solution is changed to
0.03 M sodium acetate at pH 5.4 to 5.6 and an additional
10 column volumes of eluate is collected. (The prop
erties of the coenzyme Bla compounds are such that they
are not appreciably eluted under the above conditions,
and the initial elution at pH 4.6 to 4.8 can be omitted
if desired since impurities eluted at pH 4.G arc also
eluted at pH 5.6.) The column is then eluted with 0.03
M sodium acetate at pH 6.0-7.2 whereby the coenzyme
B:3 compound is substantially completely eluted from
the column.

The primary property described herein for selection of
those fractions of the eluate which contain the ccenzyme
BU compounds is the property of these compounds of
absorbing light in the region of 260 mu, at which wave
length coenzyme B13 compounds have their maximum
absorbance. Thus, the absorbance values of the individ
ual eluate fractions collected from the chromatogram
are measured and plotted as a function of the fraction
number. On such a plot, each particular compound
elutes at maximum concentration in a particular tube,
and the concentration and the absorbance of the earlier
and later tubes are progressively lower; this necessarily
results in an absorbance "peak." The absorbance peaks
observed in this procedure may correspond to fractions
which are colorless or variously colored. Only thoss
peak fractions (using the 260 mu wave length) which
are orange or red in color contain coenzyme Bu com
pounds in substantial concentrations.

The further selection of the fractions containing co
enzyme BU compounds from amongst the red or orange
colored peak fractions is based upon the determination
of coenzyme activity using the glutamate-mesaconate
spcctrophotometric coenzyme assay and upon the deter
mination of the entire ultra violet and visible absorption
spectrum of the fractions.

The glutamate-mesaconata spcctropbotometric coea-
zyme assay is based on the observation that the rate of
formation of mesaconate from glutamate by suitable
enzyme preparations of Clostridium tetanemorphum
strain Hi is dependent upon the concentration of coen
zyme B13 compounds in the reaction mixture. Under
suitable conditions, defined below, the rate of mesaconate
formation in the assay system is substantially proportional
to the concentration of a particular coenzyme BU
compound.

"The assay depends upon the following chemical reac
tions catalyzed by suitable enzyme containing extracts of
C. tetanemorphum:

/t-metbjlaapartatt

The formation of mesaconate is detected by an increase
in ultra violet light absorption at wave lengths below
300 mu. A wave length of 240 mu is convenient to use
for this purpose. The rate of mesaconate formation is
measured by the rate of change of absorbance at 240 mu,
determined with a spcctrophotometer with silica cells
capable of measuring absorbance at wave lengths below
300 mu. The absorbance is expressed in units which
equal the log of the incident light intensity divided by
the intensity of the transmitted light.

Extracts containing enzyme suitable for the assay are

3,037,010

prepared as follows: 15 g. of cell paste of C. tetano-
morphum strain HI, freshly brvrvcsted from a 0.1 M
glutnmate —0.3% yeast extract medium, arc suspended
in 30 ml. of 0.02 M potassium phosphate buffer pH 7.6
containing 0.07 M-mercaptoclhanol. All subsequent
operations in the preparation of the enzyme extract are
carried out at 0-5" C. Approximately 3 g. of grade FFF
corundum powder and 5 g. of moist, acid-washed acti
vated charcoal Nuchar are added and the suspension is
exposed to sonic vibration in a Raytheon 10 kc. sonic
oscillator for 10 minutes at 0-5°, thereby disrupting the
cells. The suspension is then centrifuged for 10 minutes
at 16,000x# and the sediment is discarded. To 34 ml.
of the supernatant solution, 20 ml. of 1% (w./v.)
protamine sulfatc (Nutritional Bioch. Corp.) is added
slowly with mechanical stirring, in order to remove
nucleic acids. After stirring for 5 minutes the precipitate
is removed by csatrifugation at 16,000 Xg. Small aL'quots
of the clear supernatant solution, containing 20 to 25 mg.
of protein per ml., are placed in small plastic tubes and
immediately frozen. When stored nt —10* C., the
enzyme system retains much of its activity for several
months. Repeated thawing and freezing of the enzyme
solution and storage at 0* C. results in rapid loss of
activity. For this reason the enzyme solution is divided,
before feeing frozen, into small aliquots sufficient for the
assays to be performed each day.

When the enzyme extract is prepared in the absence
of charcoal, the resulting extract catalyzes both Reac
tions I and II. However, when the extract has been
treated with a suitable charcoal adsorbent either during
or after the breaking of the cells, the coenzyme BI3 com
pounds normally present in such extracts are adsorbed
by the charcoal and thus removed from the extract. Such
charcoal-treated extracts catalyze Reaction II, but they
cannot catalyze Reaction I at a significant rate unless
some coenzyme B13 compound is added.

The reaction mixture contains per ml., 0.01 M mono-
sodium L-glutamate, 0.05 M tris(hydroxymethyl)aminc-
methano chloride buffer pH 8.02, 0.01 M KC1, 0.001 M
MgClj, 0.05 ml. of a charcoal- and protamine-treated
enzyme preparation (see above) and sufficient coenzyme
to give an absorbance change of 0.01 to 0.08 unit per
minute at 240 mu corresponding to the formation of
0.0026 to 0.021 /imole of mesaconate per minute. The
reaction is started by the addition of enzyme and readings
are taken at 0.5 minute intervals for three minutes. The
rate of reaction is calculated from the change in absorb
ance during the last two minutes. The reference cell
contains sufficient mesaconate, usually about 4X10-* M,
so that the absorbance of the reaction mixture falls
between 0 and OJ.

The rate of mesaconate formation as measured by the
rate of absorbance change under the assay conditions is
approximately proportional to the concentration of
coenzyme B12 compound over the limited range indicated
above. The corresponding range of concentrations of
the coenzyme BU compound differs with different forms
of the coenzyme. With coenzyme Bla per se the useful
range is approximately 2X 10~7 M to 2x 10-« M, whereas
for benzimidazole-coenzyme B13 (whose coenzyme activity
is about 60 times that of coenzyme BU) it is approximately
4xlO-»Mto4xlO-»M.

The coenzyme activity in the glutamate-mesaconate
spectrophotometric coenzyme assay is expressed in units
of absorbance change per minute under the assay con
ditions. One activity unit is the amount of coenzyme
that causes an absorbance change of one absorbance unit
per minute. The activity unit does not have an absolute
value because the activity of the charcoal-treated enzyme-
extract differs from one preparation to another. There
fore the activity unit has a relative value which is deter
mined in relation to the activity of a standard sample
of coenzyme under identical assay conditions. A con

venient reference standard is a solution of a puriScd
sample of the bcnzimidazolc-coenzymc BI3 of knov/n con
centration, although any coenzyme sample of Iccown
concentration can be used as the standard, if desired.
5 The relative activity of the unknown sample is deter
mined with respect to the reference sample by direct
comparison in this glutamate-masaconatc spcctrcpboto-
mctric cccnzyme assay.

The glutamate-mesaconate spectrophotometric coen-
10 zyme assay is employed not only for determining
coenzyme Bi; compounds in fractions from the chromato-
gram, but is also used to assay for coenzyme Eu com
pound-activity at various stages in the purification includ
ing direct extracts of disrupted cell pasts. The
15 determination of coenzyme BU compound-activity in such
cell paste extracts provides a convenient method for
selecting microorganisms suitable for coenzyme prepara
tion. A convenient method for determining extractablc
coenzymc activity in disrupted microorganism cell pasts

20 is to suspend 50 mg. of the cell paste in 1 ml. of 0.01 M
sodium acetate buffer, pH 6.0 and heating the suspension
in a boiling water bath for 5 minutes. Tne mixture is
rapidly cooled to 0' C. and centrifuged for 5 miautcs
at 16,OOOX£ and aliquot of the color supernatant solution

25 is then assayed in the glutamate-mesaconate spectrophoto
metric coenzyme assay.

In unfractionated extracts of microbial cells, compounds
are sometimes present that cause a non-specific absorbance
change in the glutamate-mesaconate spectrophotometric

30 coenxymc assay. The presence of such compounds may
be detected and a suitable correction found by doinj a
control assay in which glutamatc is omitted from the assay
mixture. An additional correction should also be made
for the small absorbance change that sometimes occurs

35 jn the absence of added coenzyme.

As noted hereinabovc, this gluamatc-mesaconate spcc-
rro-photometric coenzyme assay facilitates the selection of
those cluate fractions from chromaiograns which con
tain coenzyme Bla compounds. The further selection of
0 eluate fractions containing coenzyme B13 compounds in a
state of high purity is achieved by determining the ap
parent spscific-coenzyme-activity of the active fractions.
By "spccific-coenzyme-activity" is meant the ratio of the
activity as determined on a particular sample divided by

45 the absorbance of that sample at 260 mu. Since the ab
sorbance at 260 mu is determined by certain impurities as
well as by concentration of coenzyme B12 compounds, the
ratio of activity to absorbance (specific-cocnzyme-aciiv-
ity) gives a quantitative measure of the purity of the coen-

50 zyme BJ3 contained in the fraction; thus, fractions having
a constant value for specific-coenzyme-activity can be as
sumed to be free of inactive impurities absorbing at
260 mu.

The specific coenzyme activity provides a convenient

55 method for characterizing coenzyme BU compounds and
is particularly effective for distinguishing between the
benzimidazole coenzyme Bla and coenzyme BU, since the
specific activity of the former is approximately 60 times
that of the latter.

80 The fractions are further characterized by determina
tion of the ultra violet and visible absorption spectrum
in order to establish whether the coenzyme BU com
pound (demonstrated in the previous tests to be present
in such fractions in relatively pure form) is the desired

55 coenzyme BU compound. The spectra of the coenzyme
BU compounds are similar in lacking the prominent ab
sorbance peak in the 350-367 mu region which is char
acteristic of all previously known vitamin BU compounds.
While the spectra of the coenzyme BU compounds are

70 generally similar, they can readily be distinguished from
one another. For example, the adenine-coenzyme BU
differs markedly from the benzimidazole-coenzyme BU
compounds in having a prominent absorbance maximum
at 458 mu, whereas the benzimidazole-coenzyme BU

75 compounds have a comparable absorbance maximum at

xnno

con T

3,037,016

about 520 mu. Also, the spectrum of bcnzimidazole-co-
enzyme Bla has an inflection at 280 mu, whereas the spec
trum of coenzyme By (containing 5,6-dimcthylbcnzimid-
azolo) has an inflection at 287 mu.

The homogeneous fractions from the column contain- 5
ing a pure coiczyme Bla compound arc now combined,
desalted by extraction into a phenolic solvent and rccx-
tractcd back into water to give a salt-free concentrated
aqueous solution of the pure coenzyme B|3 compound.
Such a solution may be used in the preparation of the 10
crystalline coenzyme either by slow evaporation or by
addition of acetone or other organic solvent in which the
ccenzyme BU compound is relatively insoluble. Alter
natively, this solution can be used directly as a substan
tially pure form of the coenzyme Bt3 compound for nu- 15
tritional purposes or for metabolic studies.

In accordance with the foregoing procedure, and utiliz
ing vitamin Bu-activity producing Schizomycctes in con
junction with a bcnzimidazolc compound as precursor,
there are obtained vitamin Bl3-active coenzyme Bj- com- 20
pounds containing a bcnzimidazole or similar nucleus, as
for example coenzyme Bu (which contains the 5,6-di-
methylbenzimidazolc) benzimidazole-ccenzyme B!a, 5-hy-

in admixture with pharmaceutical carriers or as fc."xl sup
plements in admixture with pharmacologically Lcccpted
feed additives, and the like.

The coenzyme B13 compounds produced in accordance
with this invention differ from the vitamin B13 compounds
by contaijuE3 an adcnine moiety in addition to the bctcro-
cyclic bass attached to ribosc in the vitamin B13 com
pounds. The spectra of these coenzyme BU compounds
differ greatly from the spectra of the corresponding 3l:
vitamins by having a peak with the highest extinction co
efficient at approximately 260 mu, and by lacking the
prominent peak with a high extinction coefficient in the
350-367 mu region which is characteristic of all of the
previously kaov/n vitamin Bu compounds. The coen
zyme BU compounds are readily decomposed by expo
sure to visible light or by exposure to cyanide ion. Either
of these treatments results in progressive and finally com
plete loss of ccenzyme activity. Either exposure to light
or to cyanide ion causes removal of adenine or an adcnine
derivative from the coenzyme.

A comparison of some properties and certain structural
features of coenzyme BU. benzimidazole coenzyme 3i;,
and vitamin Bu are set forth in the following table:

CoenzymeBu

Bcnzlmldazolc-
Coonzyme B"

Vitamin Bu

Mu.

Ef-««

Mm.
mu

2!™."

Mai. ... •( H
mu *!«•.

Absorption Spectrum Ic
0.03M NaAc pH 8.7

35.5

261

301-305

31. 1
12.8
12.3

278 16. 0

315
33W37

12.8

12.5

361 27.3

Inflections -. .

376
623

9.9
7.4

376
620

8.1)
7.6

&60 8.0

287
440-H5
600

280
MCM<5
600

Components:

Moles per Atom of Cobalt

1
1

1
0

l,w£I,~700"""~I

1 7

0
1
1

1
0

I.SOM.'TOO"""""

100

1
0
1

1
0.

1.3C8.
None.

+.
Converted to dl-
cyano form.

CN slowly lost.

6,6-Dtmethyl-benil-

Rlbone

Phosphate
Cyanide .

ChniBo at pH 4.8 ...
Molcculnr Welsbt

Ocbromonos Malhamen-
sis Activity

.{.

Stability In Cyanide So
lution.

Stability In Lljht

Converted to dicy-
Qnocobalamln.
Coenzyme activ
ity lost. Adeline
removed.

Converted to
hydroiocotmla-
mtn or very simi
lar epd. Coen-
ryme activity lost.
Adcnine removed
(oe derivative).

Converted to dl-

cyanobenzlmldo-
zole cobomldo.
Coeczyme activ
ity lost. Adenine
removed.
Converted to
bydrotobenzlml'
dazole cobamlde,
or very similar
cpd. Coenzyme
activity lost. Ade
nine removed (as
derivative).

droxy-benzimidazole-coenzyme B:=) 5-amino-bcnzimid-
azole-coenzyme BU, 5-nitro-beazimidazole-coenzyme BI3,
5-methyl-benzimidazole-coenzyme B;3. and the like.
Other vitamin Bu-active coenzyme Bj3 compounds con
taining heterocyclic compounds other than the benzimid-
azoles attached to the ribose may be likewise produced
utilizing vitamin Bu-activity producing Schizomycetes
in conjunction with the appropriate heterocyclic com
pound as precursor. These vitamin B13-active coenzyme
BU compounds may be administered for their nutritional
effect as such or in the form of their solutions in phar
macologically accepted liquid diluents, such as water, or

EXAMPLE 1

A culture medium for the production of the benzimid-
azole-coenzyme BU is prepared as follows: A sterile 20
liter Pyrex bottle is filled with 14 liters of distilled water
at 35-37* C. To this are added 4 liters of sterile solution

70 A and 700 ml. of sterile solution B, the compositions of
which are described hereinbelow, 200 ml. of sterile 4 M
glucose and 200 ml. of 10~J M benzimidazole. The bot
tle is then rotated to mix the contents, 0.6 g. of dry (non-
sterile) sodium hydrosulfite (Na-^OJ is added^and the

75 contents again mixed. *

"" ' r

3,037,016

Sterile Solution A. — Preparation end Sterilization .

Basamin (Anhcuscr Busch yeast extract) g— 500

Accent (monosodium glutamatc) g— 2700

MgSO4, 2 M ml— 80

FcSO,, 0.2 M ml._ 32

MnCI3. O.I M ml.. 16

NajMo04, 0.1 M ml— 16

CoCK. 0.1 M ml— 32

Cud,, 1 M— ml— 16

Distilled water to 32 liters.

To sterilize, place 4 liters of this solution in a 6-liter flask
and sterilize for 45 minutes at 18 Ib. steam pressure.

Sterile Solution B. — Preparation and Sterilization

KH.PO< (reagent grade) — g— 170

K2KPO,-3HjO (reagent grade). g— 1,200

Distilled water to 5.6 liters.

dry to recover the coenzyme completely. The final vol
ume is about 2 liters.

The solution is adjusted to pH 8.5 with 2 N NaOH
and passed by gravity flow through a 15 cm. high x 3.5

5 cm. diameter column of an iinion-cxchangc resin contain
ing quaternary ammonium groups, 8% cross-Jinked in the
hydroxide ion form (Dowcx-2, 50-100 mesh; Dow Chem
ical Company). This requires about 3 hours. The col
umn is washed with water and the combined effluents

10 pH 9.7 arc neutralized with 90 ml. of 1 M acetic acid to
pH 6.3. The volume is approximately 2.2 liters.

Phenol extraction. — Each liter of solution is extracted
with 120 ml. of 90% (w./v.) phenol-water, then twice
with 40 ml. of phenol-water. The phases art; separated

IS by ccntrifugation. The phenol phase (120 ml.) is washed
twice with 20 ml. water. The wash water is reextractcd
with 4 ml. phenol, the water is discarded and the phenol
extracts are combined. To 125 ml. of phenol phases are
added 375 ml. of ether, 125 ml. of acetone and 10 ml. of

20 water. The mixture is shaken and centrifuged to separate
the aqueous phase. The organic phase is reextractcd twice
with 10 ml. of water. The combined aqueous phase from
2.2 liters of Dowex-2 treated solution is extracted three
times with 5 ml. of ether to remove phenol and is aerated

To sterilize, place 700 ml. of this solution in a liter flask
and sterilize for 45 minutes at 18 Ib. steam pressure.

This medium is then inoculated with 750 ml. of an ac
tively fermenting pure culture of a vitamin BIJ- activity
producing strain of Clostridium tetanomorphum (strain
HI which produces vitamin B12 active compounds when

grown in a medium containing 5,6-dimethylbenzimidazolc) 25 with nitrogen to remove ether,
prepared by inoculating 5 ml. of a semisolid agar (0.2%) Chromatographic purification.— The aqueous solution

culture of the bacteria into solution A supplemented with from the phenol extraction operation (volume approxi-
0 05% of cysteine-HCl neutralized to pH 7 and incubat- mately 70 ml.) containing the partially purified benzinida-
ing this for 18-24 hours at 37' C. The 20-liter bottle is zole-coenzyme B13, is acidified to pH 3.2 with 7 ml. of 1 N
then filled with distilled water and incubated at 35-37' C. 30 aqueous hydrochloric acid solution. Since thc calculated
until the culture reaches maximum turbidity indicating salt concentration is about 0.09 M, the solution is diluted
maximum growth. This usually requires from 15-20 ^-fold to give a final salt concentration below 0.02 M;
hours, depending on temperature and condition of the l°w" salt concentrations favor adsorption of the coen-
inoculum. and corresponds to a reading of 20 to 25 zVme on thc «sin. The acidified and diluted solution is
(2-log g=0.6 to 0.7) on an Evelyn colorimeter using 35 passed into a resin column prepared as follows: 2 M so-

1S.O mm. O.D. tubes and a 540 mu (green) filter after
correcting for the absorbance of the uninoculated me
dium.

The bacteria are then harvested by centrifugation at
20,000 x 8- The 3-4 g. of cell paste per liter of medium
ihus obtained were placed in a wide mouthed polyethyl
ene container, immediately frozen in a Dry Ice-alcohol
mixture and stored at or below —10' C. until the isola
tion-purification procedure could be performed. The fore-

dium phosphate-phosphoric acid buffer pH 2.5 is allowed
to pass under gravity through a column of a suifonic acid
type cation-exchange resin which is a copolymcr of styrene
and divinylbenzimidazole containing free suifonic acid
groupings, 2% cross-linked, 200-400 mesh (Dowcx-50W,
200-400 mesh; 2% cross-linked; Dow Chemical Co.),
which is initially in the acid form, until the effluent has
the same pH as the added buffer. The resin is then
washed with distilled water until thc effluent is free from

ml. fractions are collected throughout the elution and
their absorbance at 261 mu measured. The elution of
the benzimidazole-cocnzyme BU which begins after ap-

uwu^ywi u n*«uwu K* w*» ««* *- *.«**• v« «*• j*w* *w« ••*•*•• » »*v »«» **- „ . . . • • •• •

goinc operation is carried out repeatedly to produce a 45 phosphate. The washed resin, in the mixed sodium ion-

total" of 3 86 kg. of cell paste. hydrogen ion form, is used to make a 1 cm. diameter x 80

An 83% ethanol extract is prepared from 1 kg. of cells «m- W] column- Durin8 PassaBe >"to the =olumr- thc

at a time. Two liters of absolute ethanol arc added to benzim.dazolc-cocnzyme B,, is adsorbed on the resin.

1 kg. of thawed cell paste and the mixture is homogenized ^^l™1"." thcn cluted m% approximately 800 ml.

for 30 seconds in a large Waring Blendor. The resulting 50 £0.03 M fotora aeetjto at PH 5.2 followed by about

suspension is poured into 2 liters of boiling 95% ethanol. 1500ml. of 0.03 M sodium acetate pH 6.2; individual 16
Thc mixture is heated to boiling, allowed to stand at this
temperature for 15 minutes and then filtered while hot
through a layer of diatomaceous silica on 2 large Biichner

funnels. The residue on the filter is sucked dry and 65

through the column, and is completed when approximately

750 ml. of this buffer has passed through the column, is
recognized by the appearance of an intensely reddish-
orange color and by the appearance of a prominent and
00 rather symmetrical 261 mu absorbance peak. TTre
homogeneity of the coenzyme material eluted in the peak
fractions is determined by comparing the coenzyme ac
tivity, as measured by the glutamate-mesaconate spectro-
photometric coenzyme assay, with the absorbance of thc
05 various fractions; this is expressed as relative specific
activity and is substantially constant for the central por
tion of the peak containing 80-90% of the total ab
sorbance.

The peak fractions, which have (as noted) essentially
The clear solution is adjusted to pH 7 and passed by 70 constant specific activity, and which possess the type of
gravity flow through a 15 cm. high x 3.5 cm. diameter spectrum characteristic of the coenzyme Bla compound
column of a resin copolymer of styrene and divinyl ben- containing benzimidazole attached to ribose, are combined
zene containing suifonic acid groups, 12% cross-linked, in to give a total volume of approximately 280 ml. This
the sodium ion form (Dowex 50, 50-100 mesh; Dow solution contains approximately 53 umolcs of ben-
Chemical Company). The column is washed and sucked 75 zamidazolc-cocnzyme BU (as determined by absorbance

rcsuspended in 2 liters of 80% ethanol. The suspension
is filtered as before. The combined filtrates from 3.86
kg. of cells are combined and concentrated in vacuo to
about 800 ml. To remove residual ethanol, 1.5 liters of
distilled water is addcd and the solution is again concen
trated to about 1 liter.

The resulting solution is turbid and a slimy precipitate
forms after the solution is frozen and thawed. To remove
the precipitate, which tends to clog the ion exchange resin
columns subsequently used, 10 ml. of 1 M ZnSO4 and 20
ml. of 1 N NaOH are added per liter of solution and
after standing 5 minutes thc resulting precipitate is re
moved by filtration through diatomaceous silica.

xcno

COl'v

3,037,016

measurements at 261 mu and the estimated molar extinc-
tion coefficient of 35.5X 10" cm.Vmole at 261 mu), and
in substantially pure form as indicated by the absorption
spectrum.

Tho solution containing the benzimidazole-coenzyme 6

trum very similar to that of hydroxocobalamin in the re-
gion between 320 mu and 600 mu and showing maxima
at 350-355, 410 and 525 mu.

EXAMPLE 2

scribed hereinbclow, and sufficient sterile solution of so-

residual ether. The deep red aqueous solution, contain-
icg approximately 48 umoles of coenzyme in a volume of
S ml. is placed in a vacuum desiccator over concentrated
sulfuric acid as a desiccant The desiccator is evacuated
and the solution is allowed to concentrate at 3° C. to a
volume of approximately 2 ml. during a period of several
days. During concentration of tho solution, crystalliza-
tion of the coenzyme occurs. The mother liquor is do-
canted, the crystals adhering to the walls of the container
are washed first with 90% acetone, then with 100%
acetone, and finally with ethyl ether. After removal of
ether in a vacuum desiccator there is obtained approxi-
mately 50-70 mg. of substantially pure crystalline ben-
zunjdazole-coenzyme B,a m hydrated form.

The benzimidazole-coenzyme BU forms prismatic crys-
tals having some diamond-shaped and some rectangular
faces. Tho crystals are conspicuously dichroic, being
either light yellow or deep red or a mixture of these colors.
The diamond-shaped faces appear either yellow or red,
depending on the angle of observation: the rectangular 35
faces appear red. The absorption spectrum of an aqueous
solution of this crystalline benzimidazolc-coenzyino B12
is substantially identical with the absorption spectrum of
the peak column fractions from which tho crystalline ma-
ferial is obtained. .The specific coenzyme-activity of the 40

tents

Sterile Solution A. — Preparation and Sterilization
Basamin (Anheuser Busch yeast extract) _____ g__ 500

Glucose ------------------------------ g — 4800

MgSO<, 2 M ------------------------ ml __ 80

FeSO«, 0.2 M ------------------------- ml __ 32

MnClj, 0.1 M ------------------------- ml __ 16

Na2MoO«, 0.1 M ---------------------- ml __ 16

CoCl2, 0.1 M ------------------------ ml__ 32

CaCIj, 1 M -------------------- ______ ml __ 16

Distilled water to 32 liters.

To sterilize, place 4 liters of this solution in a 6-liter flask
Md stcrilize for 45 ^^ at Jg !b> 3team ^

Sterile Solution 3. — Preparation and Sterilization

KHjPO4 (reagent grade) _g 170

K2HPO4-3HiO (reagent grade) _I"III"II__gII 1,200
Distilled water to 5.6 liters.

?Iacer70? ml" of ** *°lutlo° * a Lter flask
and stenljze for 4S minutes at 18 Ib. steam pressure.

Sterile Suspension C. — Preparation and Sterilization

fractions.

The ultra violet and visible absorption spectrum of
crystalline benzumdazole-coenzyme Bj2 hydrate (prepared
in accordance with the foregoing procedure) dissolved in
water showed maxima at 261, 315, 375, and 250 mu and
the corresponding

values were 175, 68, 53, and 39; the spectrum had in-
flection points at 280, 440-445, and 500 mu. The cobalt
content of this crystalline benzimidazole-coenzyme Bu
hydrate was found to be 3.1% corresponding to an ap-
parent molecular weight of approximately 1900. This
value includes an unknown amount of water of crys-
tallization; the molecular weight of the anhydrous crys-
tallino coenzyme, calculated on the basis of 1 mole of
cobalt, is estimated to be within the range 1500-1700.
Analysis of the benzimidazole-coenzyme Blz showed that
it contains per mole of cobalt approximately one mole
each of benrimidazole, adenine, ribose and phosphate.

Treatment of the benzimidazole-coenzyme Bj2 with 0.1
M KCN for approximately 30 minutes at room tempera-
tare results in complete loss of coenzyme activity and
results in the formation of a compound which in the
region between 300 mu and 650 mu has an absorption
substantially identical with that of the dicyano form of
vitamin BU with absorption peaks at 304 mu, 367 mu,
416 mu, 540 mu, and 579 mu.

Exposure of the benzimidazole-coenzyme BU to visible
light, such as a 100 w. tungsten filament lamp at a dis-
tance of 1 foot for a period of 30 minutes results in vir-
tually complete loss of coenzyme activity and results in
the formation of a compound having an absorption spec-

This medium is inoculated with 750 ml. of an actively
fermenting pure culture of said vitamin Bjj-activity pro-
45 ducing strain of Propionibacterium shermanii prepared
ty inoculating 5 ml of a culture of the bacteria into solu-
t'on A supplemented with 0.05% cysteine-HCl and in-
cubating this culture for 48-72 hours at 30* C The 20-
liter bottle is then filled with distilled water and incubated
at 30* C. with stirring for a period of approximately 3-10
i12^- Th* pH of ^ nisdi"™ is determined at frequent
""ervals and the pH is adjusted to pH 7, as required, by
^ ^dition of a solution of 2 N NaOH. In order to
obtain more abundant growth, additional amounts of
Slucose are added at intervals as this compound is used
u^' ^ *^e end °' tne fermentation period, the cells are
harvested by centrifugation and are obtained as a moist
**^ P35*6 ^at can ^ "^ immediately for the prepara-
^on °* coenzyme BJJ or, if desired, can be frozen and
stored for lar use.

are carried out in the dark or in very

^our tilograms of cell paste of Propionibacterium
shermanii (obtained in accordance with the foregoing

65 Procedure are extracted with approximately 21 liters of
80-90% ethanol at boiling temperature. The filtered
extract is concentrated in vacuo (45-50* C.) to remove
alcohol. The resulting aqueous solution (volume 1800
«!•) is adjusted to pH 7 with 2 N NaOH passed through

JQ a 2 cm. diameter x 36 cm. high column of a sulfonic
acid type cation-exchange resin (Dowex-50, 8x, 20-^40
mesh) in the sodium ion form. The solution is then
adjusted to pH 9.4 by addition of 2 N NaOH and is
passed through a 2 cm. diameter x 30 cm. column of a

75 quaternary ammonium type anion-exchange resin (Dow-

3,037,010

ex-2) in the hydroxide form. The combined effluent and
washings (pH 9.8) are neutralized with glacial acetic acid
to pH 6.2.
Phenol extraction. — The resulting solution (volume ap-

epproximately 30 minutes at room temperature results
in virtually complete loss of coenzyme activity and results
in the formation of a compound which in the region be
tween 300 mu and 650 mu has an absorption spectrum

proximately 4 liters) is saturated with phenol and is ex- 6 substantially identical with that of the dicyano form of

.. ,. ;.t. A < . »f nr\ar. . wlfomtTi R.. unfli aJvinmtinn DMlcs ^t T04 mu. 367 mu.

traded three times with 0.1 volume of 90% aqueous
phenol, and the coenzyme is then displaced back into
water by addition of 3 volumes of ethyl ether and 1 vol
ume of acetone for each volume of phenol. The phenol-

vitamin BU with absorption peaks at 304 mu, 367 mu,
416 mu, 540 mu and 579 mu.

Exposure of the coenzyme BU to visible light, such as
a 100 w. tungsten filament lamp at a distance of 1 foot

ether-acetone solution is extracted 3 times with 0.1 volume 10 for a period of 30 minutes, results in virtually complete

^oss °^ cocnzyme activity and results in the formation of
a compound having an absorption spectrum very similar
to that of hydroxocobalamin in the region 'between 320
mu and 600 mu and showing maxima at 350-355, 3

EXAMPLE 3

A culture of Clostridium tetanomorphum strain HI is
grown in accordance with the procedure described in Ex
ample 1 hereinabove (except that 5,6-dimethylbenzimidaz-

of water.

Chromatographic purification.— -The resulting aqueous
solution of coenzyme has a volume of approximately 600
ml. and a pH of about 7.0. The solution is acidified with

2 N HC1 to pH 3.0. The solution is diluted with distilled 15 and 525 mu.
water to 2 liters so that the final salt concentration is less

than 0.02 M. The solution is then passed into a 2 cm.
diameter x 80 cm. high column of a resin copolymer of

styrene and divinyl benzene containing sulfouic acid _r ,__-,--

groups in the mixed sodium ion-hydrogen ion form at pH 20 o|e ;n a concentration of IX 10-1 M is used in place of

3 (Dowex-50K, 2x, 200-400 mesh; Dow Chemical Co.). tne benzimidazole used in Example 1). The cells from
The column is eluted successively at 3' C. with water 2 liters of fermented broth thus produced are harvested
(250 ml.) 0.03 M sodium acetate pH 5.5 (3,200 ml.) and and treated in accordance with the phenol extraction and
0.05 M sodium acetate pH 6.4 (3,600 ml.). The eluate is chromatographic purification method set forth in de-
collected by means of an automatic fraction collector; 25 tail in Example 2 hercinabove, with suitable adjustment
each 10 minute fraction has a volume of approximately

25 ml. The coenzyme begins to elute after approxi
mately 1 liter of pH 6.4 buffer has passed through the
column. The elution of the coenzyme from the column
is recognized by the appearance of a large absorbance
peak at 260 mu, by the intense orange-red color of the
eluate, and by the presence of coenzyme activity as in
dicated by the glutamate-mesaconate spectrophotometric

coenzyme assay. The product is collected in approxi-

for the smaller quantities of starting material (approxi
mately 8 g. cell paste — %0o of the amount used in Ex
ample 2). The final aqueous solution obtained by com-
'bining the peak fractions from the elution of the coen
zyme from the resin column, followed by extraction into
phenol and displacement back into water, contains ap
proximately 0.1 pmole of coenzyme BU, as determined
from the absorbance of its solution at 520 mu and the

_ estimated molar extinction coefficient of 7.55x10* cm. V

mately 40 fractions having a total volume of about 1 liter. 35 moje>
From the 260 mu absorbance values and the estimated jjj'e identity of the coenzyme Bt2 obtained in this ex-

molar extinction coefficient of 35.5 X 10* cm.Vmole, the ample, with that obtained in Example 2 utilizing Propion-
total quantity of coenzyme B12 compounds in the peak {bacterium sharmanii, is shown by the observation that
fractions is estimated to be approximately 500 pinoles, tjjey },ave (fae same absorption spectra; the same relative
The coenzyme activities of the peak fractions are de- 40 specjfic activity in the glutamate-mesaconate spectro-
termined by the spectrophotometric coenzyme assay and photometric coenzyme assay; undergo the same spectral
the relative specific activities of the fractions calculated changes and loss of coenzyme activity when exposed to
from these activities and the absorbance measurements cyanide ion or to light; and possess approximately 1 mole
at 260 mu on the respective fractions. The ultraviolet eac]j of adenine, and 5,6-dimethylbenzimidazole per atom
and visible absorption spectra of selected peak fractions 45 of cobalt

are also determined. On the basis of the specific activity Various changes may be made in carrying out the prcs

determinations and absorption spectra, the peak frac
tions with uniform properties are selected and combined.
The resulting coenzyme solution is extracted into phenol
by the method described hereinabove and is displaced 50
back into water by the addition of ether and acetone as
also previously described. The aqueous solution thus
obtained is extracted several times with ether to remove
phenol and is then concentrated in vacuo. The final solu
tion, containing approximately 400 ^moles of coenzyme 65 structure like the cyano-benzimidazole-cobamides but
BU in a volume of about 40 ml., is deep red in color. The lacking a cyano group and possessing an adenine moiety
coenzyme BU is crystallized by further concentrating
the aqueous solution and allowing it to stand at 3* C. in

ent invention without departing from, the spirit and scope
thereof. In so far as these changes and modifications are
within the purview of the annexed claims, they are to
be considered as part of my invention.

Having thus described my invention, what I claim and
desire to secure by Letters Patent is:

i. A coenzyme BU compound having a molecular
weight within the range of about 1500 to 1700, a chemical

the dark; or, alternatively, by adding to each volume of

attached to the corphyrin portion of the molecule; char
acterized as being converted to the corresponding dicyano-
benzimidazole-cobamide with removal of adenine and loss.

aqueous solution approximately 6 volumes of acetone and 60 of coenzyme activity on treatment with cyanide ion and
approximately 3 volumes of ether until the solution be- to the corresponding hydroxo-benzimidazole-cobamide-
comes slightly turbid and allowing the resulting mixture like compound with removal of adenine and loss of activ-
to stand at 3* C. until the coenzyme B12 crystallizes. The ity on exposure to light; characterized by the ultra violet
crystals are washed with 90% acetone, then with 100% and visible absorption spectrum of its solution in 0.03 M
acetone, and finally with ether, and the ether evaporated 65 sodium acetate pH 6.7 as exhibiting a peak with the high-
in vacuo to produce substantially pure crystalline co- est extinction coefficient at approximately 260 mu and

lacking a prominent peak in the 350-367 mu region; and
when in crystalline form being further characterized as
forming prismatic crystals soluble in water, metbanol,
70 etbanol, and phenol, and substantially insoluble in ace
tone, ether and chloroform; said compound being further
characterized as supporting the growth of the microor
ganism Ochromonas malhamensis and as having coenzyme
activity as measured by the glutamatemesachonate spec-

enzyme BU in hydrated form.

The solution of coenzyme BU in .03 M sodium acetate
pH 6.7 shows absorption maxima at 260, 315, 335-337,
375 aiid 520-523 mu and the corresponding molar exten
sion coefficients (XlO* cm.Vmole) of 35.5, 12.8, 12.5,
9.9 and 7.5, respectively. Analysis of the coenzyme BU
showed that it contains per mole of cobalt approximately
one mole each of 5,6-dimethylbenzimidazole and adenine.

Treatment of the coenzyme B13 with 0.1 M KCN for 75 trophotometric coenzyme assay.

3,037,016

2. The compound cocnzyme B12, an organic substance
having vitamin Bu activity and coenryme activity as
measured by the glutamate-mesaconatc spectrophotome-
tric cocnzyme assay; having a molecular weight within
the range of about 1500 to 1700 and a chemical struc
ture liks that of vitamin BI3 but lacking a cyano group
and possessing an adenine moiety attached to the cor-
phyrin portion of the molecule; characterized as being
converted to dicyanocobalamin with removal of adenine
and loss of cocnzyme activity on treatment with cyanide
ion and to a hydroxo-cobalamin-likc compound with re
moval of adenine and loss of coenzyme activity on ex
posure to light; characterized by the ultra violet and
visible absorption spectrum of its solution in 0.03 M sodi
um acetate pH 6.7 as exhibiting an inflection at 287 DIM- 15
and absorption maxima at 260 mu, 315 mu, 335-337 mu.
375 mu and 523 mu with corresponding molar extinc
tion coefficients (XlO8 cm.Vmole) of 35.5, 12.8, 12.5,
9.9 and 7.5 respectively, and as failing to exhibit a promi
nent absorption peak in the 350-W7 mu region; and when

in crystalline form being further characterized as forming
prismatic crystals soluble in water, methanol, ethanol, and
phenol, and substantially insoluble in acetone, ether and
chloroform.

3. The compound benzimidazole-coenzyme BI3, an or
ganic substance having vitamin B12 activity and coenzyme
activity as measured by the glutamate-mesaconate spec-
irophotometric coenzyme assay; having a molecular
weight within tr>: range of about 1500 to 1700 and a
chemical structure like that of the benzimidazole analog
of vitamin 8.2 but lacking a cyano group and possessing
an adenine moiety attached to the corphyrin portion of
the molecule; characterized as being converted to dicyano-
benzimidazole-cobamide with removal of adenine and loss
of cocnzyme activity on treatment with cyanide ion and
to a hydroxobenzimidazolecobamide-like compound with
removal of adenine and loss of coenzyme activity on ex
posure to light; characterized by the ultra violet and visi
ble absorption spectrum of its solution in 0.03 M sodium
acetate pH 6.7 as exhibiting an inflection at 280 mu and
absorption maxima at 261 mu, 303-305 mu, 315 mu,
375 mu and 520 mu with corresponding molar extinction
coefficients (XlO« cm.VmoIe) of 35.5, 12.8, 12.8, 9.9
and 7.5 respectively, and as failing to exhibit a prominent
absorption peak in the 350-367 mu region; and when in
crystalline form being further characterized as forming
prismatic dichroic crystals soluble in water, methanol,
ethanol, and phenol, and substantially insoluble in ace
tone, ether and chloroform.

4. Coenzyme BU as defined in claim 2 in substantially 60
purified form.

5. Benzimidazole-coenzyme Bla as defined in claim 3
in substantially purified form.

6. Coenzyme Bu as defined in claim 2 in the form of

its crystalline hydrate. 65

7. Benzimidazole-coenzyme BU as defined in claim 3
in the form of its crystalline hydrate.

8. A process for the production of a vitamin B12-active
coenzyme BU compound of the character set forth in
claim 1 which comprises growing an Ochromonas mal
hamensis and BU coenzymc-activity producing microor
ganism in an aqueous nutrient medium and disrupting the
resulting bacterial ccfls in the substantial absence of light
and cyanide ion.

9. A process for the production of coenzyme Blz of
the character set forth in claim 2 which comprises grow
ing an Ochromonas malhamensis and Bu coenzyme-ac-
tivity producing microorganism in an aqueous nutrient
medium and disrupting the resulting bacterial cells in
the substantial absence of light and cyanide ion.

10. A process for the production of benzimidazole-co
enzyme BU of the character set forth in claim 2 which
comprises growing an Ochromonas malhamensis and Bta
coenzyme-activity producing microorganism in an aque
ous nutrient medium containing benzimidazole and dis
rupting the resulting bacterial cells in the substantial ab
sence of light and cyanide ion.

11. The process of claim 9 in which 5.6-dimethyl-
benzimidazole is incorporated in the nutrient medium.

12. A process for producing coenzyme Bla comprising:
growing in an aqueous nutrient medium, Schizomycetes
organisms of the genus Propionibacterium and capable
of producing Ochromonas malhamensis-activity; and dis
rupting the resulting bacterial cells in the substantial ab
sence of light and cyanide ion.

13. A process for producing coenzyme Bu comprising:
growing in an aqueous nutrient medium, Schizomycetes
organisms of the genus Streptomyces and capable of pro
ducing Ochromonas malhamcnsis-acli\ity; and disrupting
the resulting bacterial cells in the substantial absence of
light and cyanide ion.

14. A process for producing coenzyme Bta comprising:
growing in an aqueous nutrient medium, Schizomcycetes
organisms of the genus Pseudomonas and capable of pro
ducing Ochromonas malhamensis-activity; and disrupting
the resulting bacterial cells in the substantial absence of
light and cyanide ion.

15. A process for producing benzimidazole coenzyme
BU comprising: growing in an aqueous nutrient medium,
containing benzimidazole, Clostridium tetanomorphum
organisms capable of producing Ochromonas malhamen-
jw-activity; and disrupting the resulting bacterial cells in
the substantial absence of light and cyanide ion.

16. A process for producing coenzyme Bu comprising:
growing in an aqueous nutrient medium containing 5,6-
dimethylbcnzimidazole, Clostridium tetanomorphum or
ganisms capable of producing Ochromonas malhamensis-
activity; and disrupting the resulting bacterial cells in the
substantial absence of light and cyanide ion.

No references cited.

Ann. Rev. Biochem. 1978. 47:1-33

EXPLORATIONS OF *967

BACTERIAL METABOLISM

H. A. Barker

Department of Biochemistry, University of California,
Berkeley, California 94720

CONTENTS

EARLY YEARS I

INTRODUCTION TO SCIENCE 3

POSTDOCTORAL YEARS AT PACIFIC GROVE AND DELFT 7

A SOIL MICROBIOLOGIST AT BERKELEY 1 1

EARLY EXPERIMENTS WITH RADIOACTIVE CARBON 13

SABBATICAL INTERLUDE 15

SUCROSE PHOSPHORYLASE 16

RESEARCH ON DRIED FRUIT 18

CLOSTRIDIUM KLUYVERI: FATTY ACID METABOLISM AND AMINO

ACID BIOSYNTHESIS 18

BIOCHEMISTRY OF METHANE FORMATION 2 1

TRANSITION FROM MICROBIOLOGY TO BIOCHEMISTRY 22

THE BR FACTOR 23

PURINE DEGRADATION BY CLOSTRIDIA 24

SABBATICAL AT THE NATIONAL INSTITUTES OF HEALTH 25

GLUTAMATE FERMENTATION AND B12 COENZYMES 26

LYSINE DEGRADATION BY CLOSTRIDIA AND RELATED PROBLEMS .... 29

FINAL COMMENTS 32

EARLY YEARS

I grew up mostly in California, first in Oakland till the age of 1 1, and then
in Palo Alto till I graduated from Stanford University. My parents had been
part of the western migration. My father as a young man came to California
from Maine, where he had grown up on a poor farm in a rural environment
that was attractive to a child, but held little promise of a good life for an

1
0066-4 1 54/78/070 1 -000 1 $0 1 .00

H f

2 BARKER

adult. He worked for a time as a farm hand in the San Joaquin Valley and
later taught in an elementary school for a few years, until he could save a
little money. In 1 892 he entered Stanford with the first class, but had to drop
out before graduating, for lack of funds. He returned to the public schools
as a high school teacher. Later he became principal of a high school and
eventually a public school administrator in several cities, including Oakland
and Palo Alto, where I grew up.

My mother came to Stanford from Denver and obtained an A.B. degree
in Classical Literature and an M.A. degree in Latin. She then taught lan
guages in high school for a few years until she married. I had an older
brother who was fond of literature and eventually became a professor of
English. So there was nothing in my family background that predisposed
me to a career in science with one possible exception. Both my father and
mother were very fond of the outdoors and so each summer we spent a
month or more, whenever possible, camping in the Sierras, and living a
simple and quiet life in close contact with Nature. This resulted in my
developing a considerable familiarity with plants and animals, and the
physical environment, and perhaps even more important, developing a
sense of satisfaction and accomplishment in relatively solitary activities
such as fishing, hiking, and exploring new areas; this attitude was easily
carried over to scientific work in a laboratory.

In high school I followed a rather standard college preparatory cur
riculum including mathematics, chemistry, and two foreign languages. The
scholastic standards were not very high, so I had no difficulty getting
adequate grades with little effort. One of my dominant interests in the last
two years of high school was music. I had taken piano lessons for several
years previously with only minimal results. My enthusiasm for music was
greatly stimulated at this time by contacts and a developing friendship with
a fellow student, Robert Vetfesen, who had unusual talents as a pianist, and
at the age of 14 was already giving concerts of professional quality. As a
result, I began to work hard to develop the techniques of piano playing, only
to conclude after several years that my abilities in that direction are very
limited. Although frustrating, this experience was beneficial in opening up
to me the world of music from which I have derived much pleasure.

After graduating from high school I was fortunate to be able to spend
a year (1924-1925) in Europe with my family. Most of the winter we stayed
in Dresden, which at tha't time was a center of musical activity. I studied
the piano, learned German, read classical German literature, and went to
innumerable operas and concerts of every kind, usually occupying the
cheapest seats. I remember that one of the highlights of the season was a
musical festival honoring Richard Strauss on the occasion of his 60th
birthday, during which he conducted several of his operas and ballets.

EXPLORATIONS OF BACTERIAL METABOLISM 3

INTRODUCTION TO SCIENCE

I entered Stanford in 1925 with no idea which field I would ultimately
choose as a major. Indeed, I inclined toward literary and historical subjects.
Fortunately for me, much of the curriculum for the first two years was fixed,
and I was required to take a course in general biology. I found much of the
material both novel and interesting, and I recall that I was impressed by
the enthusiasm and personalities of some of the instructors. So the following
spring I decided to take a course in systematic botany from LeRoy Abrams.
This turned out to be a good choice for me. The class was small and
informal, and .the work consisted mainly in collecting native plants in the
adjacent fields and hills and learning to identify them by reference to
Jepson's Manualof Flowering Plants of California. I soon began to appreci
ate the diversity of plants and the influence of environment on their distribu
tion in nature. My knowledge in this area was later extended by taking a
course in plant ecology and by accompanying a graduate student, Carl B.
Wolf, on a seven-week field trip throughout the American southwest during
which we collected over 5000 plants for the Stanford herbarium.

As a result of these experiences I decided, near the end of my sophomore
year, that I would like to become some type of biologist. Since I had almost
no background in the physical sciences, this decision meant that I had to
start my real scientific education almost from the beginning. On examining
the requirements for graduation in various fields, I found that I could obtain
the physical sciences background I needed and fulfill the requirements for
an A.B. degree most quickly by majoring in the School of Physical Sciences,
which provided an introduction to mathematics, physics, chemistry, and
geology. I studied all of these subjects with enthusiasm and graduated in
the summer of 1929.

On entering graduate school I still had no definite idea as to which area
of biology I should enter. So I decided to sample introductory courses in
several areas, including plant and animal physiology, protozoology, and
psychology. I found the protozoology course given by C. V. Taylor to be
particularly stimulating because the class was very small and informal,
which allowed close personal contact with an enthusiastic teacher. Also,
because the emphasis was on microscopy, micro-manipulation, and other
techniques I was able to learn something about the behavior and physiology
of protozoa. The following spring I moved to the Hopkins Marine Station
on the Monterey Peninsula, with a small group of premedical students, to
study invertebrate zoology and embryology. The instruction was excellent
and the environment was enchanting, but the most important thing that
happened to me was a conversation with a fellow graduate student, Robert
E. Hungate. He told me that he had been getting some instruction in

78
4 BARKER

microbiology from a new member of the staff, a young Dutchman by the
name of C. B. van Niel, whom he had found to be a superb teacher. On
I lungate's advice I decided to ask van Niel to accept me as a student in the
summer quarter. There was one complication. I had planned to start on a
vacation in the Sierras somewhat before the end of the summer quarter. So
I asked van Niel whether he could let nv; start the course a week early, in
order to avoid interference with my vacation plans. He was rather surprised
at this request, but the following day he agreed. I was van Niel's only
student that summer; he spent much time with me introducing the experi
ments and discussing the results. On occasion the discussions would expand
into lectures lasting an hour or two, which were presented with a clarity,
enthusiasm, and almost hypnotic intensity that made a deep impression on
me. I quickly became convinced that microbiology was a most exciting
subject. One aspect of microbiology that van Niel emphasized was the
developing knowledge and theories of the biochemistry of yeast and bac-
lerial fermentations. Most of this material was quite new to me and I soon
began to realize that my knowledge of chemistry was still insufficient to
understand these phenomena. This realization was responsible, in consider
able part, for my later decision to change my major to chemistry.

During this summer I also assisted Taylor with some experiments on the
development of starfish eggs. Taylor was committed to spend the following
academic year as a visiting professor of Zoology at the University of
Chicago, and he invited me to come along as his research assistant. Since
this provided an opportunity to see how I would like research in
protozoology and to broaden my scientific background at another institu
tion, I accepted.

At Chicago, Taylor suggested that I investigate some aspect of the con
version of active protozoa to their resting forms, or cysts, but left me free
to decide which organism to use and how to proceed. After reading the
available literature and making some trials, I decided to use the ciliate
Colpoda cucullus, which can be cultured readily in an infusion of hay and
forms cysts abundantly under appropriate conditions. Previous studies with
various ciliates had suggested that several environmental factors, including
food supply, pH of the medium, accumulation of excretion products, and
lowered O2 tension, may induce encystment; but there was little solid
evidence to support any of these suggestions. Making use of some things I
had learned from van Niel, I was able to simplify the conditions for encyst
ment by culturing the ciliates on a suspension of bacteria in a nonnutritive
medium and show that cyst formation is almost unaffected by pH or food
supply, but that it is induced by other unidentified changes in the environ
ment associated with crowding of the ciliates. These observations formed
the subject of my first scientific publication. I also investigated the nature

79
EXPLORATIONS OF BACTERIAL METABOLISM 5

of the factor in plant and animal infusions that induces the conversion of
cysts to active ciliates, and was able to show that a number of common
organic and inorganic compounds are inactive. Subsequently, Kenneth V.
Thimann and I found that an acid ether extract of hay infusion contains
much of the activity of the original extract. Thimann & Haagen-Smit (1)
later established that the activity in ether extracts is attributable to the salts
of 1-malic and other organic acids.

While at the University of Chicago I also made some observations on the
effect of moisture on the survival of Colpoda cysts exposed to high tempera
tures. Like bacterial spores, the cysts become more heat resistant when their
moisture content is decreased. This led me to wonder whether the relation
between moisture and heat resistance of living organisms could be at
tributed to an effect of moisture on the stability of cellular proteins. A
search of the literature turned up only a few observations on this subject
and so I decided this might be a suitable project to investigate later for a
Ph.D. thesis. This could presumably be done in a chemistry department,
where I could also increase my meager knowledge of chemistry in prepara
tion for a career in microbiology. To do this I needed to find a sponsor who
would accept me as a graduate student to work on this project. Fortunately,
with the help of C. V. Taylor, I was able to persuade James W. McBain of
the Stanford Chemistry Department to do so.

Before starting graduate work in Chemistry, I again spent the summer
at the Hopkins Marine Station, this time as an assistant to J. P. Baumberger
of the Stanford Department of Physiology. One of my duties was to serve
as a teaching assistant in a small laboratory course in general physiology,
and another was to investigate the toxicity of cyanide for the brine shrimp,
Artemia salina. This remarkable creature had previously been observed to
be relatively insensitive to cyanide, and I confirmed the fact that it can swim
all day in a brine solution containing 50 mM KCN. However, the organism
becomes sensitive to cyanide when the pH of the solution is lowered. By
systematically varying the pH and the cyanide concentration we were able
to conclude that the toxicity is determined primarily by the concentration
of undissociated HCN. These observations were never published, but they
were reported by Baumberger at a local scientific meeting.

That summer Leonor Michaelis spent several weeks at the Hopkins
Marine Station as a visiting professor and I shared a small laboratory with
him. Most of his time was spent either revising the manuscript of his book
on oxidation-reduction potentials, or looking at the spectra of various dyes
and natural pigments by means of what now seems like a rather primitive
spectroscope. He also gave a few lectures on topics such as the theory and
practice of electrophoresis. These were models of organization and clarity
and greatly stimulated my interest in these areas of science. I bought his

books on mathematics, physical chemistry for students of medicine and
biology, and hydrogen ion concentration and studied them with en
thusiasm. At a later time I applied for a postdoctoral position with Michae-
lis at the Rockefeller Institute, but nothing was available.

I spent the following two years (1931-1933) working on my Ph.D. thesis,
taking the required chemistry courses and examinations, and serving as a
teaching assistant in a general biology course. For my thesis research I
decided to use egg albumin, since Hopkins, Sdrensen, and others had devel
oped methods for the crystallization of this protein, and it had been used
in some earlier experiments on heat denaturation. I soon learned that not
all the useful information about the purification of egg albumin was to be
found in the scientific literature. My first attempt to prepare the crystalline
protein from six dozen eggs, obtained from a local store, was unsuccessful,
apparently because the eggs were too old. The only useful product was some
gold cake prepared from the yolks. A second preparation, starting with
newly laid eggs, gave the expected crystalline product in good yield.

My plan to study the relation between water content and the heat stability
of egg albumin worked rather well at first. By the application of simple
methods, I was able to get satisfactory data on the effect of relative humidity
on the rate of heat denaturation and also determine the water content of
native and denatured egg albumin as a function of relative humidity. How
ever, the interpretation of the kinetic data in terms of the chemistry of
denaturation was not at all clear, and I could not think of any way of further
elucidating the problem. McBain had given me much good advice about
methods and other technical aspects of my research, but he lacked the
background in protein chemistry to be helpful at this stage. I began to doubt
that I had been wise to choose my own thesis problem and to wonder
whether I had the ability to carry it through. But gradually I found my way
out of this gloomy mood.

McBain had several good instruments for measuring physical properties,
including a polarimeter and a mercury arc light source. Since the optical
rotation of protein solutions had been reported to increase during denatura
tion, I decided to see how this property was affected by heating egg albumin
solutions. The measurements were easy to make and the observed rotations
were relatively large, but my early results were confusing because different
egg albumin samples that initially showed the same specific rotation gave
markedly different values after being heated. Only after a period of consid
erable frustration did I discover that by carefully standardizing the experi
mental conditions and having only a single variable I could get reproducible
results. This was a valuable lesson that I never forgot. The explanation of
my initial difficulties was that the specific rotation of the denatured protein
was determined not only by the time and temperature of heating, but also
by the initial pH and the protein concentration of the solution.

EXPLORATIONS OF BACTERIAL METABOLISM 7

POSTDOCTORAL YEARS AT PACIFIC GROVE
AND DELFT

I completed my Ph.D. thesis in the depths of the Great Depression and was
fortunate to get a National Research Council Fellowship for a period of two
years (1933-1935) to extend my biological training at the Hopkins Marine
Station. Since I had previously acquired an interest in both marine organ
isms and microbiology I decided to attempt to isolate some marine diatoms
and dinoflagellates and learn something of their physiology and metabo
lism. In the course of a year I was, in fact, able to isolate pure cultures of
two species of diatoms and three species of small photosynthetic dinoflagel
lates, and to maintain several other species of dinoflagellates in species-pure
culture. The diatoms were used for studies of photosynthetic quotients by
means of Warburg's manometric techniques that I learned from van Niel
and from Robert Emerson, who spent the summer at the Marine Station.
The main conclusion derived from my experiments was that diatoms, like
green algae, produce carbohydrates, rather than fats, as major products of
photosynthesis. The dinoflagellate cultures were used mainly to determine
environmental conditions favorable to the growth of these little-known
organisms. The optimum temperature of the photosynthetic dinoflagellates
was 1 8°C. This required them to be grown in a refrigerated bath that was
illuminated by tungsten lamps that generated considerable heat. The result
was an electricity bill for culturing these organisms that strained the very
modest budget of the Marine Station. Partly for this reason, I terminated
my studies of photosynthetic organisms and took up an investigation of the
utilization of organic substances by a colorless alga, Prototheca zopfii, which
van Niel had brought from Delft.

I found that Prototheca is an essentially aerobic organism that utilizes a
large number of fatty acids and alcohols, and a few sugars, as substrates for
respiration, but can also convert glucose to D-lactic acid in the absence of
oxygen. While investigating the ability of cell suspensions to oxidize various
substances, using manometric methods, I made the unexpected observation
that the quantity of oxygen consumed was much smaller than that calcu
lated for complete oxidation, .With ethanol or acetate, for example, O2
uptake was about 50% of theoretical, and with glycerol, the value was 29%.
Carbon dioxide production was also very low, which indicated that a large
fraction of each substrate was converted to other products. Since relatively
little organic material accumulated in the medium, a major part of each
substrate must have been assimilated in some form within the cells. An
analysis of all the data I had collected on O2 uptake and CO2 production
led to the conclusion that the assimilated material, corresponding to 50-
80% of the substrates, had the empirical composition of a polysaccharide.
This study directed the attention of microbiologists to the quantitative

82
8 BARKER

importance of synthetic processes that are coupled with the aerobic degra
dation of organic substrates by cell suspensions of microorganisms. This
so-called "oxidative assimilation," which is basically similar to the oxidative
conversion of lactate to glycogen by muscle, was later shown by others to
be a conspicuous feature of the aerobic metabolism of many bacteria and
yeasts (2).

During my second year at the Hopkins Marine Station I had to consider
what I would do when my fellowship ended. The job situation was very
bleak. I applied for two positions in the Departments of Food Technology
and Plant Nutrition at the University of California, but the prospects of
their being funded for the following year were poor. At the same time, I
answered an advertisement for a position as Junior Microbiologist in a US
government laboratory, only to be informed that since I had taken only one
course in microbiology I was not qualified. Fortunately, I also applied to
the General Education Board of the Rockefeller Foundation for another
fellowship to spend a year in the Delft Microbiology Laboratory with A.
J. Kluyver, and in this I was successful. After spending a few weeks in the
Sierra, my wife and I set off for Holland in July 1935.

Before leaving Pacific Grove I decided that I would like to investigate the
anaerobic degradation of glutamate and the biological production of me
thane. These were both topics that I had learned about from van Kiel. At
that time a good deal was known about the bacteria responsible for several
types of carbohydrate fermentation, but only a few observations, mostly
with mixed cultures, had been made on the anaerobic degradation of amino
acids by bacteria, van Niel thought that such studies should be done with
pure cultures and that bacteria preferentially using particular amino acids
could probably be obtained from soil or similar sources by the enrichment
culture method. He had started anaerobic enrichment cultures using vari
ous single amino acids as energy sources and found that glutamate was a
particularly good substrate. One of his students isolated a clostridium from
a glutamate medium, but was unable to carry the work further; so I inher
ited the problem.

van Niel had done no experimental work on biological methane forma
tion, but he had developed an ingenious hypothesis for the origin of me
thane, based mainly on the earlier experiments of Sohngen. The latter had
shown that carbon dioxide is reduced to methane when molecular hydrogen
is used as a second substrate and had also found that methane is the only
hydrocarbon formed from a variety of organic substrates, irrespective of the
number of carbon atoms they contain, van Niel concluded that in all these
processes the organic substrate is oxidized to carbon dioxide and water, and
this oxidation is coupled with the reduction of part of the carbon dioxide
to methane. I decided to look for further evidence in support of this hypoth-

EXPLORATIONS Oh BACTERIAL

esis, and to attempt to isolate cultures of methane-forming bacteria, which
had not been done previously.

When I first discussed these problems with Kluyver, he was sympathetic
but, I think, a little skeptical that I could make much progress on either
one during the year. He suggested that while getting started on these
problems I should isolate a bacterium fermenting tartaric acid and possibly
other C4-dicarboxylic acids, and investigate the chemistry of the degrada
tion of these compounds by the method developed in the Delft laboratory,
namely, quantitative determination of the fermentation products. In fact,
the isolation of a tartrate-fermenting strain of Aerobacter aerogenes, which
could also ferment fumarate and 1-malate, proved to be easy, and within a
few months I had data on the fermentation products. The data were inter
preted to mean that the dicarboxylic acids undergo an oxidation-reduction
reaction to give succinate and an oxidized product, probably oxaloacetate,
that is decarboxylated to pyruvate; the latter is presumably converted to
various GI and C2 products characteristic of Aerobacter aerogenes by reac
tions previously observed or postulated in other systems. No effort was
made to detect the postulated intermediates or enzymes. This was consid
ered not only too difficult, but also unnecessary for the purpose of establish
ing the pathway of the fermentation. Since the postulated pathway was
consistent with the observed yields of fermentation products and since some
of the component reactions had been demonstrated previously in other
biological systems, we felt safe in assuming, without further evidence, that
the postulated reactions occurred in these bacteria.

I made a similar study of anaerobic glutamate degradation by first isolat
ing a clostridium, later identified as Clostridium tetanomorphum, that is
capable of utilizing glutamate as a major energy source, and then determin
ing the amounts of each product formed. I finally proposed a hypothetical
sequence of reactions that might account for the observed products. The
latter were ammonia, carbon dioxide, hydrogen, acetate, and butyrate, and
the hypothetical pathway involved a more or less simultaneous deamination
and decarboxylation of glutamate to form crotonate. Crotonate could pre
sumably undergo reduction to butyrate and a coupled oxidation, by way of
^-hydroxybutyrate and acetoacetate, to acetate and hydrogen. Again, no
confirmatory evidence for the postulated pathway was obtained. As I later
found, the pathway is incorrect in almost every detail for glutamate degra
dation by C. tetanomorphum. However, other investigators (3-5) have
shown that Peptococcus aerogenes and other nonsporulating bacteria de
grade glutamate by a pathway similar to that originally postulated for the
clostridial fermentation. Although my study of glutamate fermentation did
not contribute to knowledge of intermediary metabolism, it was useful in
establishing the possibility of using single amino acids as energy sources for

10 BARKER 84

anaerobic growth, and it eventually led to the discovery of an enzymatically
active form of vitamin BI2.

While working on the degradation of C^dicarboxylic acids and gluta-
mate, I also began to search for a way of testing van Kiel's CO2 reduction
theory of methane formation with an organic substrate. Obviously what was
needed was an organic compound that could be oxidized incompletely by
methane-forming bacteria without producing carbon dioxide. The reduc
tion of carbon dioxide to methane, if it occurred, could then be observed
directly. A search of the literature turned up a short article by Omeliansky
(6) which reported that a mineral medium containing ethanol and calcium
carbonate, when inoculated with rabbit dung and incubated in the absence
of O2, undergoes a fermentation that produces gas containing mostly me
thane plus a little carbon dioxide. The high methane content of the gas
suggested that ethanol was being oxidized only as far as acetic acid.

On the basis of this report, I started an enrichment culture for methane-
producing bacteria under the conditions described by Omeliansky, but
using an inoculum of sewage sludge, and soon obtained crude cultures that
utilized ethanol rapidly according to the equation

2CH3CH2OH + CO2 -> 2CH3COOH + CH4.

The cultures were also shown to oxidize butanol to butyric acid, and the
latter to acetic acid, both reactions being accompanied by a disappearance
of carbon dioxide and the formation of an approximately equimolar quan
tity of methane. These results appeared to establish the validity of the CO2
reduction theory of methane formation for these few substrates, and with
the naivete of youth I was immediately prepared to extend this concept to
methane production from all other organic compounds. This was later
found to be an oversimplification.

My observations on the methane fermentation of ethanol by enrichment
cultures yielded another result that was destined to have a considerable
influence on my career and the development of knowledge of fatty acid
metabolism in later years. When handling various ethanol-methane enrich
ment cultures, I became aware that some had a slightly acidic odor, attribut
able to acetic acid, whereas others developed a much stronger, rancid odor.
Steam distillation of volatile fatty acids from cultures of the latter type
yielded substantial amounts of a relatively water-insoluble liquid organic
acid that was identified as /i-caproic acid. This was always accompanied by
butyric acid. The formation of C4 and €5 fatty acids in high yields from
ethanol in an anaerobic environment was an unexpected discovery that I
reported to Kluyver with considerable excitement. Only after a careful
search of the literature did I find that in 1868 a student of Pasteur, A.
Bechamp, had observed the same phenomenon and reported the isolation
of 75 g of caproic acid from a culture containing 106 g of ethanol (7)!

EXPLORATIONS OF BACTERIAL METABOLISM 1 1

The publication of a report on this work was delayed for a year while an
industrial company, to which Kluyver served as scientific adviser, investi
gated the possibilities of using the process for the commercial production
of caproic acid. So far as I know nothing ever came of this. Nevertheless,
the company provided me with a small retainer that made it possible, the
following year, to start construction of a cabin in the mountains of Califor
nia which we still use each summer.

A SOIL MICROBIOLOGIST AT BERKELEY

Toward the end of the year in Delft I accepted an appointment as Instructor
in Soil Microbiology and Junior Microbiologist in the Division of Plant
Nutrition of the Agricultural Experiment Station, University of California.
As an instructor I at first assisted C. B. Lipman in teaching a laboratory
and lecture course in soil microbiology that was required of all undergradu
ate students in the Soil Science curriculum, and later I was given sole
responsibility for the course. Since my formal training in microbiology was
slight, and my knowledge of soil microbiology in particular was even
smaller, I had to work hard during the first years to learn enough about the
subject to teach the fundamentals and those aspects that might be of some
interest to students of soils. Fortunately, the students had reasonably good
backgrounds in chemistry and general biology, although I found that be
cause of the nature of the curriculum, they were generally more interested
in the inorganic and physical properties of soils than in the microbial
transformations of organic compounds. Since my interest was mainly in the
latter area, a few years later I developed, in collaboration with Michael
Doudoroff of the Bacteriology Department, and Reese H. Vaughn and
Maynard A. Joslyn of the Food Technology Department, a new course in
Microbial Metabolism in the Bacteriology Department that attempted to
cover the knowledge of intermediary metabolism that was rapidly develop
ing during that period. Later, Roger Y. Stanier and Edward A. Adelberg
also participated in teaching this course, which attracted graduate students
from several areas of biology.

Since I had an appointment in the Agricultural Experiment Station, I was
supposed to make some contribution to agricultural research. The chairman
of Plant Nutrition, Dennis R. Hoagland, asked me to join in the study of
a nutritional disease of fruit trees and other plants, known as "little leaf."
Shortly before my appointment, Hoagland and his associates had made the
important discovery that this disease is caused by a deficiency of zinc, and
he was actively engaged in investigating the conditions affecting the zinc
requirement. Field observations seemed to indicate that little leaf symptoms
were often particularly severe in areas, such as former corrals, that had
received large amounts of animal manure; this suggested that microorgan-

86
12 BARKER

isms are somehow involved in increasing the effect of zinc deficiency. Hoag-
land had begun to investigate this phenomenon by growing several
successive crops of corn in pots of corral soil and had found that each
successive crop grew more poorly, presumably because of increased zinc
deficiency. Finally the condition became so severe that corn seeds would
scarcely germinate. Hoagland asked me to see whether I could find any
basis, microbiological or otherwise, for this phenomenon. I tried a number
of experimental approaches, using sterilized and unsterilized soil, and soil
reinoculated with various bacteria isolated from the original soil, but they
led nowhere. Finally I made extracts of the soil to see whether they con
tained any material that would affect seed germination. It turned out that
an extract was as poor a medium for germination as the original soil, and
the explanation was that the salt concentration was just too high for corn.
This terminated my experiments on corral soils. I did some other experi
ments on the effect of bacteria on the development and minor element
nutrition of sterile plants grown in water culture but none of these produced
any readily interpretable data. So with Hoagland's approval I abandoned
research on bacteria-plant interrelations and devoted all my efforts to inves
tigating simpler microbial systems.

The facilities available for microbiological research were very modest
when I arrived in the Division of Plant Nutrition. They included an incuba
tor room, a very old autoclave that did not always develop the expected
temperature, a homemade oven for sterilizing glassware, a microscope, and
a supply of test tubes and flasks. Most of thq mechanical and electrical
instruments that are now considered indispensible for research, such as
centrifuges, colorimeters, respirometers, and pH meters, were lacking. Fur
thermore very little money was available in 1936 to purchase equipment of
any sort. I well remember asking Hoagland whether I could order a $15
Seitz filter that I needed to sterilize media. He eventually approved my
request but only after examining his budget to see whether we could afford
it.

In part because of the limited facilities, my students and I initially con
centrated on the isolation of various interesting kinds of anaerobic bacteria,
which could be done with the available supplies. The bacteria included
Methanobacterium omelianskii, the organism apparently responsible for
the conversion of ethanol and carbon dioxide to acetate and methane;
Clostridium kluyveri, responsible for the formation of butyric and caproic
acids from ethanol; Clostridium acidi-urici and Clostridium cylindro-
sporum, which decompose uric acid and other purines; Streptococcus
allantoicus, which degrades allantoin anaerobically; Clostridium tetano-
morphum and C. cochlearium, which ferment glutamate; Clostridium pro-
pionicum and Diplococcus glycinophilus, which utilize alanine and glycine,

87
EXPLORATIONS OF BACTERIAL METABOLISM 13

respectively; and Butyribacterium rettgeri and Clostridium lactoaceto-
philum, which ferment lactate in different ways. These organisms provided
many of the biochemical problems I was to investigate in later years.

The isolation of each of the above-mentioned organisms involved some
special problems, but none was as difficult as the initial isolation of C.
kluyveri. I have already mentioned that some enrichment cultures for
ethanol-utilizing, methane-forming bacteria produce considerable amounts
of butyric and caproic acids. Microscopic examination of such cultures
showed that they always contained a large spore-forming bacterium in
addition to a smaller bacterium (Methanobacterium omelianskii) that was
apparently responsible for the formation of methane. I undertook to isolate
the spore-former by serial dilution in the same medium used for the en
richment cultures but with agar. It soon became apparent that isolated
colonies of the spore-formers could not grow in this medium, since none was
found beyond the second dilution, although M, omelianskii grew at much
higher dilutions. As it seemed possible that the inability of the spore-former
to grow in higher dilutions might result from the absence of suitable growth
factors, I tried supplementing the medium with yeast autolysate and found
that the addition of a very high level of this material would permit it to
develop, though poorly. The problem then was to distinguish colonies of the
caproic acid-forming clostridium from the many contaminating clostridia
that thrived on yeast autolysate. This was eventually accomplished by using
a remarkably sensitive but inexpensive instrument, my nose, to detect the
presence of caproic acid in individual colonies picked with a micropipet. By
these methods, I eventually isolated a pure culture of C. kluyveri but was
disappointed to find that it produced little caproic acid in a yeast autolysate-
ethanol medium. Considerable additional time and effort were required to
find that the major essential nutrient derived from yeast autolysate is acetate
and the minor nutrients are carbon dioxide, biotin, and p-aminobenzoate.
When all these compounds were supplied, C. kluyveri grew readily, deriving
energy from the conversion of ethanol and acetate to butyrate, caproate,
and hydrogen (7).

EARLY EXPERIMENTS WITH RADIOACTIVE
CARBON

I first became involved in experiments with radioactive carbon in 1939.
Through my colleague Zev Hassid I met Sam Ruben of the Chemistry
Department and Martin D. Kamen of the Radiation Laboratory, who had
begun to use HC in the study of photosynthesis and dark CO2 fixation by
higher plants and algae. Ruben was the dynamic and tireless promoter of

14 BARKER

1 'C; and he was always interested in finding new biological systems to which
the isotope could be effectively applied. When I pointed out that the carbon
dioxide reduction theory of methane formation from organic compounds
could be tested with "C02, he was eager to collaborate.

Our experiments on the fermentation of ethanol by M. omelianskii con
firmed the earlier conclusion that methane is derived from carbon dioxide
and further demonstrated a considerable incorporation of carbon dioxide
into cellular materials. An experiment on the fermentation of methanol by
a Methanosarcina species was less convincing; although a small incorpora
tion of carbon from carbon dioxide into both methane and cell material was
observed, the results were not sufficiently quantitative to permit an unam
biguous interpretation. This was a serious limitation of "C as a tracer. The
21-min half-life allowed only about 4 hr to prepare the "COi, set up the
experiment, carry out the incubation, separate the products, and prepare
and count the final samples. The time was generally insufficient to get more
than semiquantitative data. Despite this limitation we were later able to
obtain useful data on the incorporation of carbon dioxide into acetate
during the fermentation of purines by C. acidi-urici and into the carboxyl
groups of propionic and succinic acids during fermentations by propionic
acid bacteria.

The more complicated experiments with UC always involved a group
effort. In order to reduce the duration of an experiment to a minimum it
was necessary to plan every step of the preparative and analytical proce
dures ahead of time, and to make a dry run to be sure that everything
necessary was available and working. Since our group had the lowest pri
ority for use of the cyclotron, the actual experiments were always done at
night and frequently could not be started before 1 AM. After the incubation,
everyone was busy for a while carrying out some part of the separation
procedure. Then we gathered about Ruben in the early hours of the morn
ing to watch the counting of the samples. There was always a sense of
excitement and drama when the incorporation of CO2 into some metabolic
product was shown by the high speed ticking of the counter. We felt that
science was really progressing!

Carbon 14 was first prepared in significant amounts by Ruben and Ka-
men in 1 940 (8), but because of wartime restrictions and the untimely death
of Ruben, the isotope did not become available for experimental purposes
until 1944. At that time T. H. Norris of the Chemistry Department and I
recovered the 14C from several hundred liters of saturated ammonium
nitrate solution that had been exposed to stray neutron radiation from the
60-inch cyclotron. This was a messy job lasting several days. It involved
boiling aliquots of the solution in a 12-liter flask, passing the vapors through
a condenser and over hot copper oxide, and then absorbing the CO2 in alkali

EXPLORATIONS OF BACTERIAL METABOLISM 15
89

and precipitating it as BaCO3. My share of the product was 1.8 g of
BaCO3 that had a rather low specific activity of about 1.5 X 105 cpm per
mmole. This amount, small by current standards, proved to be sufficient for
several fairly complicated tracer experiments on bacterial metabolism.

Although by this time I had some experience with tracer methodology,
I knew virtually nothing about the technical aspects of estimating radioac
tivity because Ruben had previously always done the counting on a home
made counter that only he could operate. Fortunately for me, just about the
time 14C became available Kamen lost his position in the Radiation Labora
tory because of wartime hysteria aroused by his conversation with a Russian
consular official, and he was able to collaborate with me on the first tracer
experiments with I4C. He taught me the art of making mica window Geiger
tubes and many other tricks of tracer methodology, and I in turn con
tributed something to his education in microbiology and biochemistry. It
was a most useful and pleasant collaboration.

We first examined the role of carbon dioxide in the fermentation of
glucose by Clostridium thermoaceticum. This bacterium had been shown to
ferment glucose and xylose with the formation of over 2 moles of acetic acid
per mole of sugar. The high yield of acetic acid, and the virtual absence of
carbon dioxide or other one-carbon product, suggested that part of the
acetic acid was formed from carbon dioxide. This hypothesis was shown to
be correct by fermenting glucose in the presence of 14CO2 and establishing
that the isotope is incorporated into both carbon atoms of acetate, and that
over 2 moles of carbon dioxide are actually formed and reutilized during
the fermentation. Similar experiments showed that Butyribacterium rettgeri
also uses carbon dioxide and converts it to acetic and butyric acids during
the anaerobic degradation of lactate.

A somewhat more elaborate tracer experiment on the conversion of
ethanol and acetate to butyrate and caproate by C. kluyveri provided sub
stantial evidence that acetate, or a compound in isotopic equilibrium with
acetate, is an intermediate in the conversion of ethanol to €4 and Q fatty
acids, and that caproic acid synthesis almost certainly involves the addition
of a C2 unit to the carboxyl carbon of butyrate rather than the reciprocal
reaction (7). The latter conclusion was later confirmed by showing that
I4C-labeled caproic acid derived from [l-I4C]butyric acid and ethanol is
labeled almost exclusively in the /8-carbon atom.

SABBATICAL INTERLUDE

In 1941 1 became eligible for my first sabbatical and was fortunate to receive
a fellowship from the Guggenheim Foundation. I spent the first two months
with L. F. Rettger at Yale University studying the fermentation products

90
16 BARKER

and cultural characteristics of various nonsporulating anaerobic bacteria,
which included an organism we later called Butyribacterium rettgeri. The
last two months were spent with W. H. Peterson at the University of
Wisconsin learning methods that had been developed there for investigating
bacterial nutrition and assaying for growth factors by microbiological meth
ods. The remainder of the year was spent with Fritz Lipmann in the
Surgical Laboratories of the Massachusetts General Hospital. I had been
attracted to Lipmann by his studies of enzymatic pyruvate oxidation by
Lactobacillus delbrueckii, and by his stimulating review on phosphate bond
energy. When I arrived he was engaged in the isolation of the labile phos
phate compound formed from pyruvate that was soon shown to be acetyl
phosphate. Lipmann determined the phosphate content of the isolated
product and I contributed to its characterization by estimating the acetate
content.

Before working with Lipmann all my research had involved the use of
living bacteria, either as growing cultures or as cell suspensions. He intro
duced me to methods of preparing and studying cell-free extracts, and to
techniques of detecting and estimating intermediate metabolites by colori-
metric and other relatively sensitive procedures. The method that Lipmann
favored for making bacterial extracts consisted of simply drying cells in a
vacuum desiccator over ¥2®$ a°d then extracting them with buffer. This
seems primitive by comparison with currently available methods, but it was
inexpensive and served well for a number of later studies of bacterial en
zymes at Berkeley.

SUCROSE PHOSPHORYLASE

On returning to Berkeley I continued to study various bacterial fermenta
tions, some of which have already been mentioned, and also became in
volved in two new lines of research: a study of enzymatic sucrose
degradation and an investigation of the deterioration of dried fruit during
storage.

The investigation of sucrose degradation was initiated by Michael
Doudoroff. He had isolated an H2-oxidizing bacterium that also utilized a
wide range of organic substrates. An interesting peculiarity of this organ
ism, Pseudomonas saccharophila, was that it oxidized sucrose more rapidly
than the component monosaccharides, glucose and fructose. About the time
I returned from sabbatical leave Doudoroff came to the conclusion that
further analysis of this phenomenon could only be made by the use of cell
extracts. At my suggestion he made some dried cell preparations and soon

EXPLORATIONS OF BACTERIAL METABOLISM 17

found that suspensions of the dried cells in a sucrose-phosphate solution
caused a rapid esterification of inorganic phosphate. To identify and quanti-
tate the products he enlisted the cooperation of Nathan O. Kaplan, who had
had experience with the characterization of phosphate esters during his
thesis research with David M. Greenberg, and W. Z. Hassid, who was a
carbohydrate chemist. Together they demonstrated that the major en
zymatic reaction is an apparently reversible conversion of sucrose and
orthophosphate to fructose and glucose- 1 -phosphate. Because Hassid,
Doudoroff, and I often had lunch together, and the conversation frequently
dealt with the sucrose problem, I was gradually drawn into this research
and contributed in various ways to the planning of the experiments and the
isolation and characterization of sucrose and other disaccharides that can
be synthesized by the phosphorylase from appropriate substrates (9). My
most significant contribution to this research came as a result of an experi
ment that Doudoroff and I had planned to investigate the incorporation of
32P into glucose- 1 -phosphate under various conditions. We incubated glu
cose- 1 -phosphate and 32Pj with sucrose or fructose expecting that the re
versible enzymatic reaction would result in the formation of labeled
glucose- 1 -phosphate. Almost as an afterthought we included a control with
only glucose- 1 -phosphate and 32Pj, and were surprised to find that more
32P was incorporated into glucose- 1 -phosphate in the absence of the sugars
than in their presence. In fact we did not believe the first result, and
concluded that there had been a mix up of the samples. However, repetition
confirmed the initial observation. We discussed the result for some time and
by the next day reached the conclusion that the simplest interpretation was
a reversible reaction of glucose- 1 -phosphate with enzyme to form a cova-
lently bonded glucosyl enzyme and Pj. This soon led to the idea that sucrose
was probably reacting in a similar way with the enzyme to form glucosyl
enzyme and fructose. This in turn implied that the glucosyl moiety derived
from sucrose could be transferred to another glucosyl acceptor such as
sorbose to form glucosidosorboside in the complete absence of inorganic
phosphate. Although I do not now recall the exact course of the discussion
leading to these conclusions, I think that Doudoroff, who had a very agile
mind, was the first to sense the probable explanation of our results. In any
event, with Hassid's collaboration we were soon able to demonstrate the
predicted synthesis of disaccharides by glucosyl transfer from sucrose in the
absence of phosphate (10). These results established the concept that su
crose phosphorylase functions as a glucosyl-transferring enzyme, and pro
vided substantial, though indirect, evidence for the existence of a covalent
glucosyl enzyme compound, which was demonstrated many years later by
Voet & Abeles(ll).

92
18 BARKER

RESEARCH ON DRIED FRUIT

Like many Americans in the early 1940s I felt an urge to assist in some way
in the great conflict in which the nation was engaged. So in 1943 I eagerly
accepted the invitation of my friend Emil M. Mrak of the Department of
Food Technology to participate in a Quartermaster Corps project on meth
ods of retarding the deterioration of dried fruit during storage, particularly
since the work could be done on the campus and would not preclude other
research activities. The project provided funds for an assistant; I was fortu
nate to select Earl R. Stadtman, a graduate of the Soil Science program who
had taken my course in soil microbiology and later had assisted me in
growing Chlorella on a large scale for Ruben. At first we knew almost
nothing about the problems of preparing and storing dried fruit and soon
discovered that the scientific literature dealing with these subjects was very
meager. Mrak introduced us to the conventional methods of handling dried
fruit, and then Stadtman and I, and later Victoria Haas, undertook a
systematic study of factors influencing the deterioration of dried apricots.
This required first the development of a reasonably quantitative measure of
quality. Since fruit darkens progressively during storage this was accom
plished by visually comparing the color of an alcoholic extract of fruit with
a series of standards. We then proceeded to determine the effects of temper
ature, moisture, sulfur dioxide, and oxygen, and their interrelationships, on
storage life, which was defined as the time required to reach an arbitrary
degree of darkening (12). Several effects were revealed that had not previ
ously been observed, or at least not adequately appreciated. Our results did
not help to shorten the war, since they were not published until after its
conclusion. I hope they have had some beneficial effect on the quality of
commercial dried fruit, but I do not know that this is so.

CLOSTRIDIUM KLUYVERI: FATTY ACID
METABOLISM AND AMINO ACID BIOSYNTHESIS

After the war Earl Stadtman decided to do his Ph.D. thesis with me and
undertook to explore the enzymatic reactions participating in the energy
metabolism of C. kluyveri. He soon found that crude extracts of dried cells
are able to catalyze the anaerobic conversion of ethanol and acetate to
butyrate and caproate, as well as the aerobic oxidation of ethanol and
butyrate. This exciting discovery opened up the possibility of identifying the
enzymatic reactions involved in the oxidation and synthesis of fatty acids.
In fact the analysis of the system progressed rapidly. Stadtman found that
acetyl phosphate is a product of the oxidation of both ethanol and butyrate
in a phosphate buffer, and is an essential substrate for the synthesis of

EXPLORATIONS OF BACTERIAL METABOLISM iy

butyrate when hydrogen is used as a reducing agent. Other significant
findings were the discovery of an acetyl-transferring enzyme (phosphotran-
sacetylase) and an enzymatic system for using acetyl phosphate to activate
other fatty acids. Later, in Lipmann's laboratory, Stadtman and his asso
ciates showed that both of these enzyme systems require CoA and catalyze
the formation of acyl-CoA compounds (13, 14).

Investigation of the utilization of several C4 compounds that had been
postulated to be intermediates in the reversible conversion of butyrate to
acetate and acetyl phosphate established that acetoacetate can be either
reduced to /3-hydroxybutyrate or cleaved to acetyl phosphate and acetate,
and that vinyl acetate can undergo a dismutation forming butyrate, acetyl
phosphate, and acetate. However, tracer experiments showed conclusively
that neither acetoacetate nor vinyl acetate could be an intermediate in
butyrate oxidation or synthesis. Since no other C4 compound at the oxida
tion levels of /S-hydroxybutyrate and acetoacetate was used in this system,
and no intermediate accumulated in sufficient amounts to be detected by the
available methods, we were forced to the conclusion that the intermediates
must be relatively stable complexes of C4 compounds with a coenzyme or
other carrier. This interpretation, which I first presented in a lecture before
the Harvey Society in May 1950, was developed during discussions with
Stadtman, and later with Eugene P. Kennedy, who spent a year with me
as a postdoctoral fellow.

The following year I was invited to give a major lecture on the formation
and utilization of active acetate at the first Symposium on Phosphorus
Metabolism at Johns Hopkins University. I am not sure why I was selected
for this assignment, although it was probably connected with the fact that
Lipmann and Ochoa, who were major contributors to this area of research,
were regarded at that time as competitors, and someone thought that selec
tion of a neutral third party would be more diplomatic. In any event, I felt
a great responsibility to present a comprehensive and balanced review of the
whole field, covering the work that had been done with animal as well as
bacterial systems. This required a major effort; I spent several months
studying the literature and trying to arrive at a unified interpretation of the
often incomplete and sometimes conflicting experimental results. Finally I
reached the conclusion that acyl-CoA compounds are not only primary
products of the oxidation of pyruvate and acetaldehyde, and primary sub
strates in the synthesis of acetoacetate and citrate, as had already been
demonstrated, but that they must also be intermediates in the oxidation and
synthesis of butyrate (15). I proposed a pathway for butyrate oxidation to
acetyl phosphate via butyryl-CoA, vinylacetyl-CoA, ,8-hydroxybutyryl-
CoA (by implication), acetoacetyl-CoA, and acetyl-CoA that was very
similar to that later demonstrated experimentally by Lynen and others.

94
20 BARKER

Vinylacetyl-CoA was postulated to be the initial oxidation product of
butyryl-CoA because vinyl acetate is used more readily than crotonate by
extracts of C. kluyveri. This apparently results from the specificity of the
CoA transferase in this organism. Robert Bartsch later found that C.
kluyveri contains a special isomerase that converts vinylacetyl-CoA to
crotonyl-CoA.

Another aspect of the metabolism of C. kluyveri that proved to be of
interest was the biosynthesis of its amino acids. Since C. kluyveri could be
grown in a medium containing ethanol, acetate, and carbon dioxide as the
only carbon compounds, other than small amounts of biotin and p-
aminobenzoate, it was apparent that the cellular amino acids must all be
synthesized from C2 compounds and carbon dioxide. Tracer experiments by
Neil Tomlinson showed that about 25% of the cellular carbon was derived
from carbon dioxide and 75% from acetate. Examination of the 2-, 3-, and
4-carbon amino acids derived from the proteins of bacteria gro%vn in the
presence of 14CO2 or [1-14C] acetate established that the amino acid car-
boxyl groups are derived from carbon dioxide and the a-carbon atoms are
derived from the carboxyl carbon of acetate. The results were consistent
with the interpretation that C. kluyveri carboxylates acetyl-CoA and pyru-
vate to form pyruvate and oxaloacetate and then converts these compounds
into the indicated amino acids. The postulated carboxylation reactions were
subsequently demonstrated in C. kluyveri by Stern (16).

Tomlinson also investigated the origin of the carbon atoms of glutamate
in C. kluyveri and found, in contrast to what had been observed in other
organisms, that the a-carboxyl and ,8-carbon atoms are derived mainly
from the carboxyl carbon of acetate, and that the y-carboxyl carbon atoms
are derived mainly from carbon dioxide (17). He pointed out that these
results could be accounted for by the usual reactions for the conversion of
oxaloacetate and acetyl-CoA to glutamate, provided the aconitase in C.
kluyveri had an unconventional stereospecificity resulting in the formation
of a double bond in ciy-aconitate between the central carbon atom and the
methylene carbon atom derived from oxaloacetate. This change in the
position of the double bond would cause a reversal of the positions of the
glutamate carbon atoms derived from oxaloacetate and acetate, as com
pared to glutamate formed by the usual tricarboxylic acid cycle reactions.
This plausible hypothesis was eventually disproved by Gottschalk, who
obtained convincing evidence that the citrate synthase, rather than the
aconitase of C. kluyveri, displays an atypical stereospecificity. He found that
C. kluyveri contains an (R)-citrate synthase rather than the (S)-citrate
synthase characteristic of most other organisms. The (R)-citrate synthase
of C. kluyveri fully accounts for the unusual origin of the carbon atoms of
glutamate. This type of citrate synthase apparently occurs in only a few
anaerobic bacteria (18).

EXPLORATIONS OF BACTERIAL METABOLISM 21

* OH
HOOC-CH2T ..CH2COOH

COOH [l-'4c]glu

* / (S)-Citrate

HOOC-CH, 0

-t- CH3CO-CoA
COOH

HOOGCH^ .CHgCOOH

•"V1 '

COOH — — [5-l4c]glu

(R)-Citrote

BIOCHEMISTRY OF METHANE FORMATION

Since our earlier tracer experiments with nC on the origin of methane in
the fermentations of methanol and acetate had given equivocal results,
when I4C became available I decided to reinvestigate these problems. The
immediate stimulus for this was a report by Buswell & Sollo (19) showing
that little 14C is incorporated into methane when unlabeled acetate is fer
mented in the presence of 14CO2- This result was dearly contrary to the
COi reduction theory, but it did not specifically identify the source of
methane carbon. I therefore encouraged Thressa Stadtman to study the
fermentation of specifically labeled acetate; her results showed that virtually
all the methane carbon is derived from the methyl group of acetate (20).
She also established that methanogenic bacteria convert methyl alcohol to
methane by a process not involving carbon dioxide reduction. In a further
effort to define the chemistry of the conversion of acetate to methane Martin
J. Pine investigated the fermentation of acetate labeled in the methyl group
with deuterium, and found that the methyl group is incorporated as a unit
into methane without loss of attached hydrogen or deuterium. The fourth
hydrogen atom was shown to come from the solvent. These results appeared
to exclude an oxidation-reduction of the methyl group during methane
formation, although the possibility that the same hydrogen atoms are re
moved and returned to the methyl carbon cannot be entirely eliminated.
Disregarding this possibility, the results of the various tracer experiments
are consistent with a simple decarboxylation of acetate to methane and
carbon dioxide. However, this still seems unlikely since it is difficult to
imagine how an organism can obtain useful energy from such a process. As
yet no one has succeeded in obtaining a cell-free extract with which to make
a further analysis of the chemistry of the conversion of acetate to methane.
In 1956 I undertook to summarize the results of our studies on methane
fermentation and to correlate this with the contributions of other groups.

96
22 BARKER

This led to the proposal of a generalized pathway for the formation of
methane from either acetate, methanol, or carbon dioxide, ail of which are
known to be used by some methane-forming bacteria (20). The main fea
tures of this pathway were the carboxylation of an unspecified carrier and
the sequential reduction of the carboxyl group to a methyl group that was
finally converted to methane. The methyl groups of acetate and methanol
were postulated to enter this sequence by a more or less direct methyl
transfer to the carrier and be either reduced to methane or oxidized to
carbon dioxide by a reversal of the carbon dioxide reduction pathway, or
both. This conceptual scheme seems to have been of some value to later
students of methane fermentation (21).

TRANSITION FROM MICROBIOLOGY
TO BIOCHEMISTRY

Since my original position at Berkeley was that of a soil microbiologist and
I ended up as a biochemist, I should mention some of the stages of my
metamorphosis. I remained a member of Plant Nutrition until 1950. At that
time, following the death of D. R. Hoagland, its long-time chairman, five
members of the faculty — Zev Hassid, Paul K. Stumpf, Eric E. Conn, Con
stant C. Delwiche, and I — whose interests were primarily biochemical,
formed a new Department of Agricultural Biochemistry in the College of
Agriculture. When the Biochemistry and Virus Laboratory was completed
in 1951 we moved in along with the new Biochemistry Department and the
Virus Laboratory. Although the laboratories were an improvement over
those we had previously occupied, the administrative arrangements in the
building were difficult for several years because of an almost constant
struggle over authority and space. This situation was greatly ameliorated
when Esmond Snell became chairman of the Biochemistry Department.
Shortly thereafter Hassid and I transferred into that department, and the
other members of Agricultural Biochemistry moved to the Davis campus
of the University to establish a new, and now flourishing, Department of
Biochemistry and Biophysics. In 1964 the remaining interdepartmental
problems were resolved by moving the Biochemistry Department to a new
building.

From 1936 to 1948 my students obtained advanced degrees in the gradu
ate curricula of Bacteriology, Microbiology, or Agricultural Chemistry.
The Biochemistry Department at Berkeley during that period was part of
the Medical School; graduate degrees in biochemistry were not available to
students studying with other faculty members. Since many students in other
departments were doing research on biochemical problems and wished to
be recognized as biochemists, there was considerable interest among both

students and faculty in setting up an academic mechanism for giving de
grees in biochemistry outside of the Biochemistry Department. I. L. Chai-
koff of the Physiology Department and I took the lead in organizing an
interdepartmental group major, called Comparative Biochemistry, to take
care of this problem. A curriculum for a Ph.D. degree in Comparative
Biochemistry was approved in 1948 and from then until 1958, when I joined
the new Biochemistry Department, most of my students majored in this
field. I took on the responsibilities of graduate student adviser in Compara
tive Biochemistry when the group was organized, and retained the position
until my academic retirement in 1975. During this period about 75 students
obtained Ph.D. degrees in Comparative Biochemistry. Subsequently many
of these students have contributed substantially to the world of biochemis
try; notable examples of graduates from the earlier years of this program
are Elizabeth F. Neufeld, Paul A. Srere, and Earl Stadtman.

THE BR FACTOR

I have previously mentioned some experiments on Butyribacterium rettgeri,
an anaerobic bacterium that catalyzes butyric acid fermentation of lactate
and carbohydrates. In 1950 one of my students, Leo Kline, tried to grow
the organism in a synthetic medium and found that it required a small
amount of yeast extract in addition to the then known nutrients and growth
factors. An examination of some properties of the essential material, called
the BR factor, established that it was a very stable carboxylic acid, readily
extractable with organic solvents from acid aqueous solutions; in addition,
it occurred in several more complex forms that were not soluble in organic
solvents until released by vigorous acid or alkali hydrolysis. These proper
ties were similar to but not identical with those of some other unidentified
growth factors, including a Lactobacillus casei factor studied by Guirard,
Snell & Williams (22), and a pyruvate oxidation factor for Streptococcus
faecalis reported by O'Kane & Gunsalus (23). At this stage, I should have
contacted these investigators in order to make a closer comparison of the
various preparations. Instead, after Kline had completed his thesis, I con
tinued work in the isolation of the BR factor. I obtained about 100 pounds
of Penicillium notatum mycelium, a good source of BR factor, prepared
many gallons of autolyzate, acid hydrolyzed the material in an autoclave,
built a large liquid-liquid extractor, extracted the hydrolyzate for weeks,
and with the aid of an assistant, performed innumerable tedious and not
always completely reproducible assays. After some additional steps, we
obtained several hundred milligrams of material substantially purified but
still containing a number of components both active and inactive. About
this time Gunsalus visited Berkeley and in the course of conversation we

98
24 BARKER

found that the properties of the BR factor and the pyruvate oxidation factor
were very similar. By exchanging samples we found that they were in fact
identical. Since Gunsalus' preparations were considerably purer than ours,
I immediately abandoned the attempt to further purify the BR factor.
Subsequent observations demonstrated that lipoic acid is highly active as a
growth factor for B. rettgeri.

The lipoate requirement of B. rettgeri continued to be of interest because
the function of the factor appeared to be different from that in other organ
isms. Lipoate had been shown to function as an electron carrier in the
oxidation of pyruvate. Kline and others found on the contrary that B.
rettgeri does not require lipoate for the utilization of pyruvate, but only for
the utilization of lactate. Since the products formed from lactate and pyru
vate are qualitatively the same, it was concluded that lipoate probably
functions as an electron carrier in the oxidation of lactate to pyruvate.
Martin Flavin became interested in the role of lipoic acid in this system
when he was in my laboratory, and later collaborated with C. L. Witten-
berger in a study of this problem. They reached the tentative conclusion that
in lactate oxidation, enzyme-bound lipoate mediates electron transfer be
tween an unidentified electron carrier and DPN (24). Further analysis of
the specific role of the lipoate-containing enzyme in the lactate-oxidizing
system in B. rettgeri has been impeded so far by the instability of the system
(25).

PURINE DEGRADATION BY CLOSTRIDIA

From 1937 to 1957 one of my major research interests was the degradation
of uric acid and other purines by clostridia. I started on this project as a
result of a conversation with a colleague who raised chickens. He had filled
a large container with chicken droppings, which contain uric acid, and
water, and was greatly impressed by the rapid rate at which the mixture
developed a strong ammoniacal odor. I undertook the isolation of the
responsible bacteria and had no difficulty in obtaining a number of cultures
that showed a high degree of specificity for the degradation of uric acid and
a few other purines. Jay V. Beck, my first graduate student, joined me in
studying the physiology and nutrition of the bacteria, which we named
Clostridiumacidi-uriciand C. cylindrosporum. and in identifying the fermen
tation products. We found that both organisms decompose uric acid, xan-
thine, and guanine readily, and hypoxanthine more slowly, with formation
of acetate, carbon dioxide, and ammonia as major products; in addition,
C. cylindrosporum forms significant amounts of glycine. Later, Norman
Radin found that formate is also a fermentation product. Since both clos-
tridium species were able to activate glycine as a reducing agent and decom-

99
EXPLORATIONS OF BACTERIAL METABOLISM 25

pose it when uric acid was simultaneously available, glycine appeared to be
a normal intermediate in purine degradation. Various enzymes and metabo
lites known to participate in the aerobic degradation of purines were not
detected in the clostridia, and consequently we concluded that the pathway
of purine degradation in these bacteria is quite different from that in aerobic
organisms. This conclusion was strengthened by a number of tracer experi
ments on the origin of the product carbon atoms. The early experiments
showed that both carbon atoms of acetate and the carboxyl group of glycine
are derived in part from carbon dioxide. Later experiments by Jon L.
Karlsson and by Jesse C. Rabinowitz with specifically labeled purines,
glycine, and formate established a similarity between the pathways of pu
rine degradation by clostridia and of purine biosynthesis by other organisms
(26). The pieces of the jigsaw puzzle of the degradative pathway were finally
assembled into a coherent picture as a result of enzymatic studies initiated
by Radin and carried to completion by Rabinowitz. Radin found that the
first step in uric acid utilization is its reduction to xanthine, which is then
converted by crude enzyme preparations to glycine, formate, carbon diox
ide, and ammonia. Glycine can be oxidized to acetate, carbon dioxide, and
ammonia, and serine is converted by way of pyruvate to the same products.
These results, in conjunction with those of the tracer experiments, suggested
that acetate is formed mainly by the sequence glycine -> serine -» pyruvate
-»• acetate. Radin also obtained presumptive evidence for the formation of
one or more aminoimidazoles, none of which was identical with 4-amino-5-
carboxamidoimidazole, which had been implicated in purine biosynthesis.
These observations suggested that the pyrimidine ring of xanthine is ini
tially cleaved at the 1-6 bond to yield 4-ureido-5-carboxyimidazole. The
formation of this intermediate was confirmed by Rabinowitz, who then
proceeded to elucidate the further enzymatic steps in purine degradation,
including the role of folic acid, in elegant detail (26, 27). The last contribu
tion to this area of research from my laboratory was a study by Willard H.
Bradshaw of the properties, particularly the substrate specificity, of the
xanthine dehydrogenase of C. cylindrosporum, the enzyme responsible for
the reduction of uric acid to xanthine.

SABBATICAL AT THE NATIONAL INSTITUTES
OF HEALTH

As a result of my early experience with 14C, I had come to rely heavily on
the application of tracer methods to intact cells for the elucidation of
various problems of bacterial metabolism. When the use of intact cells
seemed inadequate, I occasionally encouraged my students to use cell-free
extracts, but until the early 1950s we did not attempt to purify specific

100
26 BARKER

enzymes. The stimulus to investigate individual reactions of metabolic path
ways through the use of purified enzymes was provided by Arthur Korn-
berg, who spent part of the summer of 1951 in my laboratory learning how
to handle anaerobic bacteria. He spoke with such enthusiasm about the
advantages of using purified enzymes that I decided I should get some
experience in the art of enzyme isolation. The following year I spent six
months with Kornberg at the National Institutes of Health. He and I shared
a small laboratory and I was able to draw upon his knowledge and experi
ence whenever it was required. I learned a great deal from him in a few
months that I was later able to apply to my own research.

In Kornberg's laboratory I investigated two unrelated problems. One was
the purification of the coenzyme A transferase from C. kluyveri, using an
assay method developed by Earl Stadtman. I tried every known method of
enzyme purification on this transferase, but even with Kornberg's advice I
had very little success; the best preparation was purified only about fivefold
with a 3 1 % yield. However, even the methods that did not give any purifica
tion provided valuable experience, and that was what I needed. My second
research problem, suggested by Kornberg, was the isolation and characteri
zation of ATP from the sulfur-oxidizing bacterium, Thiobacillus thiooxi-
dans, which had been reported to differ from the ATP of other organisms
by having the phosphate groups attached to the 3', rather than the 5'
position of adenosine. During the isolation of ATP I learned how useful ion
exchange resins can be for separating charged molecules, and during the
characterization of ATP I came to appreciate the value of enzymes as
specific and convenient analytical reagents. The conclusion of our work was
that the ATP of thiobacillus is the same as that of other organisms.

GLUTAMATE FERMENTATION
AND B12 COENZYMES

1 have already mentioned my early studies on glutamate degradation by
Clostridium tetanomorphum. Further investigation of the chemistry of this
process was put off for many years while I was involved in what seemed to
be more exciting problems. A stimulus to return to a study of glutamate
metabolism was provided indirectly by Kornberg, who isolated a histidine-
degrading strain of C. tetanomorphum while visiting my laboratory. My
student, Joseph Wachsman, investigated the early steps of histidine degra
dation by this organism and concluded that glutamate is an intermediate
in this process, as it is in histidine degradation by aerobic organisms. He
then studied the degradation of glutamate by both tracer and enzymatic
methods and showed that the carbon chain is cleaved between carbon atoms

2 and 3 to form acetate from carbon atoms 1 and 2, and pyruvate from

101
EXPLORATIONS OF BACTERIAL METABOLISM 27

carbon atoms 5, 4, and 3 (26). The pyruvate is oxidized to carbon dioxide
(carbon 5), hydrogen, and presumably acetyl-CoA (carbon atoms 4 and 3),
which is mainly converted to butyrate. These results established that gluta-
mate was being degraded by a novel pathway. A clue to the nature of the
pathway was provided when Wachsman identified mesaconic acid, a
branched-chain unsaturated dicarboxylic acid, as an intermediate in gluta-
mate degradation.

'COOH

H2C-NH2
I

3CH2

I
4CH

5COOH
L- Glutomate

'COOH
2CHNH

'COOH
2CH

CH3-4CH

CH3-4C

COOH

/3- Methyl
aspartate

I
COOH

Mesaconate

The nature of the carbon skeleton rearrangement in the glutamate-mesa-
conate conversion was established a little later by Agnete Munch-Petersen,
who convened [4-14Cl] glutamate enzymatically to mesaconate and deter
mined the position of the isotope in the product. The result proved that the
bond between carbon atoms 2 and 3 of glutamate is broken, and a new bond
is established between carbon atoms 2 and 4, leaving carbon atom 3 in a
methyl group. A further study of the carbon chain rearrangement estab
lished that the first product formed from glutamate is the amino acid
3-methyl-L-aspartate, which is then deaminated to form mesaconate. The
inter-conversion of glutamate and 3-methylaspartate proved to be the most
novel and interesting step in glutamate degradation. The branched-chain
amino acid was missed in the early investigations of this system because
some of its properties are very similar to those of glutamate, and because
the equilibria in the system are unfavorable for its accumulation in quantity.
It was first detected as a product of mesaconate amination only after we
found that the enzyme catalyzing its reversible conversion to glutamate can
be inactivated by treatment with charcoal.

A rather detailed account of the circumstances leading to the isolation
of the charcoal-absorbable cofactor for the mutase and its identification as
a derivative of vitamin B12 has recently been published (28) and need not
be repeated here. But perhaps a few comments may be of interest. In
retrospect, the isolation of the corrinoid coenzymes was rather straightfor
ward once we had reached an adequate level of understanding of the en
zymatic system in which it functioned. We had a specific, sensitive, and

28 BARKER

reasonably convenient enzymatic assay; the coenzyme was relatively stable
except to one environmental factor, and its physical properties were ideally
suited to permit purification by ion exchange and solvent extraction tech
niques. Nature had put only one roadblock in our way, namely, the instabil
ity of the coenzyme to light. Our failure to recognize this property caused
much difficulty and frustration during the early stages of our investigation,
which lasted almost two years. Once this property was recognized, the
isolation of the coenzyme could be completed in a few weeks. The critical
factor for the recognition of the light effect was the development of a rapid
spectrophotometric assay for the coenzyme. We should have done this
much earlier, but the advantages of such an assay were not as evident at
the time as they are in hindsight.

I cannot leave this topic without at least mentioning my associates,
students and postdoctoral fellows, who made important contributions to
the successful outcome of our work on the isolation and characterization
of corrinoid coenzymes. Agnete Munch-Petersen first undertook to purify
the coenzyme and established some of its ionic properties; Herbert Weiss-
bach first recognized the coenzyme to be a corrinoid compound and con
tributed in many ways to the identification of its structure; Harry
Hogenkamp determined the structure of the two nucleotides formed by
photolysis of the coenzyme; John Toohey established optimal conditions for
corrinoid coenzyme formation by C. tetanomorphum, and isolated and
characterized several coenzyme analogs from bacteria and liver; Benjamin
Volcani developed a bioautographic method for the identification of small
amounts of coenzyme analogs; Jeff Ladd determined the pKa values of the
coenzymes and the effect of ionization on the absorption spectra; David
Perlman of the Squibb Institute for Medical Research assisted us greatly by
providing large quantities of propionic acid bacteria containing various
coenzyme analogs; Axel Lezius identified'the major corrinoid coenzyme in
a methane-producing bacterium; Roscoe Brady demonstrated the reactions
involved in the adenosylation of corrinoid compounds in Propionibacterium
shermanii; and Robert Smyth assisted in many ways with the assay and
initial isolation of the coenzymes.

While studies of the structure of the corrinoid coenzymes were progress
ing, we simultaneously tried to learn something about the chemistry of the
mutase reaction, but with little success. Attempts to detect either free or
coenzyme-bound intermediates gave negative results, so we concluded that
they must have a very short life. A somewhat more significant conclusion
was reached in an investigation of hydrogen transfer during the mutase
reaction. Arthur lodice found that solvent hydrogen is not incorporated
into products in appreciable amounts; this supported the interpretation that
hydrogen is transferred as either H° or H~, but not as H+. After Lenhert
& Hodgkin (29) showed the presence of a deoxyadenosyl group in the

EXPLORATIONS OF BACTERIAL METABOLISM 29

103
coenzyme, Fujio Suzuki and I investigated the role of the coenzyme as a

hydrogen-transferring agent by looking for a transfer of tritium from
[3H-methyl]3-methylaspartate to coenzyme. A significant amount of
tritium was found in the coenzyme; unfortunately, the coenzyme from a
control experiment without enzyme showed about half as much tritium, so
the results were ambiguous. Not long thereafter Abeles and his associates
clearly demonstrated that the coenzyme functions as a hydrogen-transfer
ring agent by the use of synthetic, tritium-labeled coenzyme in the diol
dehydrase reaction. We later confirmed that the coenzyme functions in the
same way in the glutamate mutase reaction.

To learn more about the mode of action of glutamate mutase I felt it
would be desirable to have a highly purified preparation. This turned out
to be more complicated than I anticipated. Early attempts to purify the
activity showed that it depends on the presence of two readily separable
proteins which we called the E and S components. The relatively unstable
E component, with a molecular weight of about 125,000, was purified by
Suzuki; and the relatively stable S component, with a molecular weight of
17,000, was purified by Robert L. Switzer. Although we learned something
about the molecular and kinetic properties of these proteins and the condi
tions for their interaction, we were unable to demonstrate separate functions
for the two subunits, if such they be, or understand how they interact to
form the catalytically active species. This remains a problem for the future.

After the discovery of the role of corrinoid coenzymes in the glutamate
mutase reaction, I considered the possibility that they might also participate
in the methylmalonyl mutase reaction, but never got beyond the stage of
speculation. Soon afterward, several groups of investigators demonstrated
that the coenzyme is indeed required for this reaction. Another process in
which vitamin B12 had been implicated by the nutritional experiments of
Snell, Kitay & MacNutt (30) was the conversion of ribonucleotides to
deoxyribonucleotides in Lactobacillus leichmannii. When Raymond Blak-
ley came to my laboratory I encouraged him to see whether corrinoid
coenzymes participate in this conversion. He was able to obtain a cell-free
preparation that reduced the ribose moiety of CMP to a deoxyribose moiety
and established that the reaction is strongly stimulated by corrinoid coen
zymes. After returning to Canberra, Blakley purified the ribonucleotide
triphosphate reductase responsible for deoxyribose formation and clarified
the novel role of the coenzyme in this oxidation-reduction reaction.

LYSINE DEGRADATION BY CLOSTRIDIA
AND RELATED PROBLEMS

In 1962, Olga Rochovansky came to my laboratory as a postdoctoral fellow
and said she would like to investigate the anaerobic degradation of lysine

30 BARKER

while getting experience in handling anaerobic bacteria. The year before,
Thressa Stadtman (31) had reported that cell-free extracts of Closiridium
sticklandii are able to convert lysine to acetate, butyrate, and ammonia. She
had identified several cofactors required for the reaction, but had been
unable to detect any intermediate in lysine degradation, even when one or
another of the cofactors was omitted from a reaction solution. Since it
seemed possible that another organism might provide enzyme preparations
more suitable for detecting intermediates, after consultation with Stadtman,
Rochovansky undertook to isolate a lysine-degrading anaerobe. She suc
ceeded in obtaining such an organism (Clostridium SB4) from sewage
sludge and went on to show that the cofactor requirements for lysine
degradation by extracts are almost the same as for C. sticklandii.

The search for intermediates in lysine degradation by extracts of SB4 was
started by my student, Ernest A. Rimerm'an. We decided to begin by adding
all the known cofactors except coenzyme A in the expectation that interme
diates found before the CoA-dependent reaction might accumulate in larger
amounts than those formed subsequently. Rimerman soon found that omis
sion of CoA caused the accumulation of significant amounts of a heat-labile
neutral compound that could be separated from other products by paper
electrophoresis. This compound was identified as 3-keto-5-aminohexanoic
acid, an unexpected product to be derived from lysine, which is substituted
in the 2 and 6 positions. An explanation for the location of the carbonyl
group was obtained by Ralph N. Costilow who was visiting my laboratory.
He looked for other intermediates in lysine degradation by omitting DPN
from an otherwise complete reaction solution, and by paper ionophoresis
at neutral pH he detected a second basic amino acid that overlapped lysine.
At a lower pH this amino acid separates readily from lysine and can be
easily assayed. The new amino acid was isolated and identified as L-3,6-
diaminohexanoic acid (yS-lysine), a compound previously known only as a
component of some polypeptide antibiotics. The formation of this amino
acid indicated that the first step in lysine degradation is a migration of the
amino group from the 2 to the 3 position. This was later established more
firmly after purification of the responsible enzyme, L-lysine aminomutase,
by Thomas P. Chirpich; he also demonstrated that the enzyme is stimulated
by pyridoxal phosphate, ferrous ion, and S-adenosylmethionine. The second
intermediate in anaerobic lysine degradation, and the immediate precursor
of 3-keto-5-amino hexanoate, was soon found to be 3,5-diaminohexanoate.
This compound was first recognized by Stadtman & Renz (32); it was
independently discovered in my laboratory by Eugene E. Dekker while
looking for an intermediate accumulating in the absence of corrinoid coen
zyme. Thressa Stadtman and her associates at the National Institutes of
Health later purified and extensively investigated the corrinoid coenzyme-

105
EXPLORATIONS OF BACTERIAL METABOLISM 31

CH2CH2CH2CH2CHCOO" —=S — *- CH2CH2CH2CHCH2COO"
i i AdoMet i i

+NH3 *NH3

LYSINE /3-LYSINE

Bfi-P

B,2CoE

NAD
CH3CHCH2CCH2COCT — ^^- CH3CHCH2CHCH2COCr

1 A ' '

"""Mi-Is ° *NH3 +NH3

3-KETO, 5-AMINOHEXANOATE 3,5-DIAMINOHEXANOATE

dependent enzyme responsible for the formation of 3,5-diaminohexanoate,
whereas we concentrated on the enzymes responsible for the formation and
degradation of 3-keto-5-aminohexanoate.

The enzyme catalyzing the oxidative deamination of 3,5-diaminohexano
ate to the 3-keto acid was purified by John J. Baker and shown to be a highly
substrate-specific, but otherwise conventional, dehydrogenase. Su-Chen L.
Hong and Ing-Ming Jeng found that the degradation of 3-keto-5-aminohex-
anoate requires the presence of acetyl-CoA, but the nature of the enzymatic
reaction responsible for the degradation eluded us for some time. The
acetyl-CoA requirements suggested that the degradation would follow the
usual pathway for fatty acid oxidation; formation of a CoA thioester of the
yS-keto acid followed by a thiolytic cleavage, which in the lysine degradation
system would result in the formation of 3-aminobutyryl-CoA and acetyl-
CoA. However, numerous attempts to detect the postulated intermediates
and products were unsuccessful.

At this point we decided to switch to another experimental approach to
the problem, namely, the synthesis of the postulated 3-aminobutyryl-CoA
and the test of its ability to be further degraded. Jeng soon found that
extracts of our lysine-fermenting clostridium contain a highly active deami-
nase that converts L-3-aminobutyryl-CoA to crotonyl-CoA. The presence
of this enzyme and crotonase accounted for our earlier inability to detect
3-aminobutyryl-CoA as a product of 3-keto-5-aminohexanoate degrada
tion, but did not account for our failure to detect the other possible interme
diate, 3-keto-5-aminohexanoyl-CoA. The nature of the reaction responsible
for the removal of 3-keto-5-aminohexanoate in the presence of acetyl-CoA
was finally determined by Takamitsu Yorifuji, who purified the responsible
3-keto-5-aminohexanoate cleavage enzyme, and found to our surprise that
it catalyzes the following reaction:

CH3CHCH2COCH2CO2 + Ac-CoA ** CH3CHCH2CO-CoA + AcAcCK
NHt NHt

106
32 BARKER

This is a previously unrecognized type of reaction for the degradation and
synthesis of jS-keto acids.

Since the study of lysine degradation by clostridia had turned up several
novel types of reactions, I decided to investigate analogous enzymatic reac
tions in two aerobic bacteria that utilize yS-lysine or 3,5-diaminohexanoic
acid as an energy source. Although these investigations are not yet com
plete, studies by Henry N. Edmunds, Su-Chen L. Hong, Gerhard Bozler,
John M. Robertson, and Masahiro Ohsugi have established that the type
of /3-keto acid cleavage reaction discovered in clostridia also occurs in both
aerobic bacteria. The j8-lysine decomposing organism is of additional inter
est because it catalyzes both an initial acetylation of the substrate and a
novel but as yet not fully denned type of deacetylation reaction at a later
step in the degradation sequence.

FINAL COMMENTS

It will be obvious to the reader that the central focus of my scientific career
has been the exploration of bacterial metabolism, generally the energy
metabolism of anaerobic bacteria, with the objective of establishing meta
bolic pathways or of identifying novel enzymatic reactions. With some
exceptions this has been a relatively quiet area of science, usually peripheral
to the main stream of biochemical research, and therefore not subject to
much competition. Consequently, I have always worked in a rather relaxed
atmosphere and have been able to enjoy several weeks vacation with my
family each summer in the mountains, without developing a bad conscience
for neglecting my students or suffering a fear of being scooped.

Most of the research embodied in my publications, particularly in my
most productive years, was done by my students and postdoctoral asso
ciates. I have been fortunate in having many bright, enthusiastic, and dedi
cated collaborators, several of whom regretfully could not be mentioned in
this chapter because of limitations of space. Much that we have accom
plished is attributable to their skill and intuition.

Literature Cited

1. Thimann, K. V., Haagen-Smit, A. J. 6. Omeliansky, V. L. 1916. Ann. Inst. Pas-
1937. Nature 140 645^6 teur Paris 30:56-60

2. Clifton, C. E. 1946. Adv. EnzymoL 1- Barker, H. A. 1947. Antonie van Leeu-
6-269-308 wenhoek J. Microbiol. Serol. 12:167-76

3. Horler, D. F, McConnell, W. B, West- 8" **$!£*' D 1963' J~ Chem' EduC'
lake, D W. S. 1966. Can. J. Microbiol 9 «««, z ^^^ ^ ^^

12:1247-52 H A 1947 Arch Biochem. u:29-37

4. Johnson, W. M., Westlake, D. W. S. 10 Doudoroff, M., Barker, H. A., Hassid,
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5. Buckel, W., Barker, H. A. 1974. /. Bac- \\. Voet, J. G., Abeles, R. H. 1970. / Biol.
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EXPLORATIONS OF BACTERIAL METABOLISM

107

12. Stadtman, E. R.. Barker, H. A.. Haas,
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13 Stadtman, E. R. 1954. Rec. Chem. Prog.
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15. Barker, H. A. 1951. In Phosphorus Me
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16 Stern, J. R. 1965. In Non-Heme Iron
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18. Gottschalk. G., Barker, H. A. 1
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Proc. 26:343

108

CURRICULUM VITAE

Horace A. Barker

Professor Emeritus, Biochemistry

University of California, Berkeley

April 1982

EDUCATION

Stanford University, A.B., Physical Science, 1929
Stanford University, Ph.D., Chemistry, 1933

EMPLOYMENT

Professor, University of California, Berkeley, Agricultural Experiment Station, 1936-1959

Professor, University of California, Berkeley, Department of Biochemistry, 1959-1969

Visiting Professor, Stanford University Medical School, 1962

Chairman, UC Berkeley Department of Plant Nutrition, 1949-1950

Chairman, UC Berkeley Department of Plant Biochemistry, 1950-1953

Vice Chairman, UC Berkeley Department of Agricultural Biochemistry, 1958-1959

Vice Chairman, UC Berkeley Department of Biochemistry, 1959-1962

Chairman, UC Berkeley Department of Biochemistry, 1962-1964

Professor, Biochemist, UC Berkeley Department of Biochemistry, 1969-1975

Professor Emeritus, 1975-

MEMBERSHIPS

American Chemical Society

American Society of Biological Chemists

American Society of Biochemistry and Molecular Biology

Society of American Bacteriologists

Biochemical Society (British)

National Academy of Sciences

American Institute of Nutrition

American Academy of Arts and Science

109

HONORS, AWARDS

Sug. Res. Award of National Academy of Science, 1945

Carl Neuberg Award, American Soc. Europ. Chem., 1959

Borden Award in Nutrition, American Institute of Nutrition, 1962

Election to National Academy of Sciences, 1953

Guggenheim Foundation Fellow, 1941-42, 1962

Sc.D. (honorary), Western Reserve University, 1964

California Scientist of the Year, 1965

Hopkins Memorial Medal and Lectureship, 1 967

National Science Medal (Presidential Award), 1 969

Faculty Research Lecturer, University of California, 1972

Honorary Membership in American Society for Microbiology, 1980

31 - Biochemist, National Me...fessor Horace A. Barker dies at 93 http://www.berkeley.edu/news/media/releases/2001/01/08_barkr.html

110

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